Antibodies specific for hyperphosphorylated tau and methods of use thereof

ABSTRACT

The present invention relates to a novel class of monoclonal antibody that specifically binds the phosphorylated serine 396 residue on pathological hyperphosphorylated (PHF) tau (pS396), as well as to methods of using these molecules and their tau binding fragments in the treatment of Alzheimer&#39;s disease and tauopathies.

RELATED APPLICATIONS

This Application is a Continuation of U.S. application Ser. No.16/188,489, filed Nov. 13, 2018, which is a Continuation of U.S.application Ser. No. 15/207,836, filed Jul. 12, 2016, and claimspriority to Great Britain Application No. 1518375.9, filed Oct. 16,2015, and Great Britain Application No. 1512211.2, filed Jul. 13, 2015.The entire contents of these applications are incorporated herein byreference in their entirety

FIELD OF THE INVENTION

The present invention relates to a novel class of monoclonal antibodythat specifically binds the phosphorylated serine 396 residue onpathological hyperphosphorylated (PHF) tau (pS396), as well as tomethods of using these molecules and their tau binding fragments in thetreatment of Alzheimer's disease and tauopathies.

REFERENCE TO SEQUENCE LISTING

This application includes one or more Sequence Listings pursuant to 37C.F.R. 1.821 et seq., which are disclosed in computer-readable media(file name: 0995.txt, created on 23 Jun. 2016, and having a size of 40kB), which file is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Age-related neurodegenerative diseases such as Alzheimer's disease (AD)and dementia are one of the largest societal challenges today. The WorldHealth Organization estimates that costs for care of the elderly willcontinue to increase and that the number of diagnosed dementia caseswill triple by 2050 (World Health Organization and Alzheimer's DiseaseInternational—Status Report (2012) DEMENTIA: A public health priority,WHO). The first treatments for AD were neurotransmitter modulators suchas acetylcholine esterase inhibitors and NMDA modulators. Thesetherapies became available at the turn of the millennium and still formthe cornerstone for symptomatic relief of memory deficits related todementia and AD. However, these drugs do not target the underlyingcauses of AD, accumulation of amyloid-β (Aβ) peptide and tau proteinaggregates and associated loss of neuronal synapses and eventuallyneurons.

Longitudinal, community-wide studies of the elderly (Weiner, M. W. etal. (2014) ADNI online: http://www.adni-info.org/; Breteler, M. M. etal. (1992) Neuroepidemiology 11 Suppl 1, 23-28; Launer, L. J. (1992)Neuroepidemiology 11 Suppl 1, 2-13) together with large genome-wideassociation studies (Lambert, J. C. et al. (2013) Nat. Genet. 45,1452-1458) have shown that AD is a heterogeneous mix of dementias whereup to 10 percent of the advanced AD patients lack amyloid pathology(Crary, J. F. et al. (2014) Acta Neuropathol. 128, 755-766).Furthermore, seminal pathological studies by Braak & Braak (Braak, H.and Braak, E. (1996) Acta Neurol. Scand. Suppl 165, 3-12) demonstrated aclear correlation between the degree of neurofibrillary tangle pathologyand cognitive state prior to autopsy. These observations have beenreinforced by several investigators (Nelson, P. T. et al. (2012) J.Neuropathol. Exp. Neurol. 71, 362-381), and in recent longitudinalbiomarker studies, which indicate that cerebrospinal fluid (CSF) levelsof tau and phospho-tau increase throughout early and late stages of thedisease (Jack, C. R., Jr. et al. (2013) Lancet Neurol. 12, 207-216).

As indicated above, the microtubule-associated protein, tau, and itshyper-phosphorylated version, form the main constituent of intracellularneurofibrillary tangles, which are one of the main hallmarks of AD.Furthermore, specific genetic variants of tau are associated withfamilial forms of fronto-temporal dementia (FTD). Appearance of taupathology in AD occurs in a distinct spatial pattern, starting in theentorhinal cortex, followed by hippocampal and cortical areas (Braak, H.and Braak, E. (1996) Acta Neurol. Scand. Suppl 165, 3-12). The specificstage of tau pathology also correlates well with cognitive abilities(Nelson, P. T. et al. (2012) J. Neuropathol. Exp. Neurol. 71, 362-381;Braak, E. et al. (1999) Eur. Arch. Psychiatry Clin. Neurosci. 249 Suppl3, 14-22). Taken together, this evidence forms the basis of a tau-basedhypothesis for AD. It entails that the intracellular accumulation of tauleads to microtubule degeneration and spinal collapse. As a result,communication between neurons malfunctions and cell death follows.Recently, it has also been shown that tau itself may form anendo-pathogenic species that can transmit neurodegeneration from onecell to the next (Clavaguera, F. et al. (2009) Nat. Cell Biol. 11,909-913).

I. Tau As An Endo-Pathogen

Clavaguera and colleagues have demonstrated that tau itself may act asan endo-pathogen (Clavaguera, F. et al. (2009) Nat. Cell Biol. 11,909-913). Low spin brain extracts were isolated from P301S tautransgenic mice (Allen, B. et al. (2002) J. Neurosci. 22, 9340-9351),diluted and injected into the hippocampus and cortical areas of youngALZ17 mice. The ALZ17 mouse is a tau transgenic mouse line which onlydevelops late pathology (Probst, A. et al. (2000) Acta Neuropathol. 99,469-481). The injected ALZ17 mice quickly developed solid filamentouspathology, and administration of immuno-depleted brain extracts fromP301S mice or extracts from wild type mice did not induce tau pathology.Fractionation of the brain extracts in soluble (S1) andsarcosyl-insoluble tau (P3) (Sahara, N. et al. (2013) J. Alzheimer's.Dis. 33, 249-263) and injection of these into ALZ17 mice demonstratedthat the P3 fraction is most competent in inducing pathology. Itcontains most of the intracellular hyper-phosphorylated filamentous tau.The majority of pathology could also be induced when injecting P301Sextracts into the brains of wild type mice, but no NFTs were formed. Insubsequent studies, Clavaguera et al. have shown that human tauextracted from post-mortem brain tissue of other tauopathies(Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP),and Corticobasal Degeneration (CBD)) may also induce tau pathology inthe ALZ17 model (Clavaguera, F. et al. (2013) Proc. Natl. Acad. Sci.U.S.A. 110, 9535-9540). Since the presentation of these data, severalother tau seeding and spreading models have been reported (Ahmed, Z. etal. (2014) Acta Neuropathol. 127, 667-683; Walker, L. C. et al. (2013)JAMA Neurol. 70, 304-310). The main conclusion from these studiesindicates a mechanism by which pathogenic tau in intracellularinclusions is secreted from the cell into the periplasmic space. Thepathological tau material is then transported along the vesicular sheathin both anterograde and retrograde direction and subsequently taken upby neighboring cells by means of bulk endocytosis. This mechanismexplains why the spread of pathology observed in human disease follows adistinct anatomical pattern. Intriguingly, peripheral administration ofpathological tau may accelerate the formation of tau pathology in ALZ17mice (Clavaguera, F. et al. (2014) Acta Neuropathol. 127, 299-301). Thisspreading mechanism may explain disease propagation in otherproteinopathies (Goedert, M. et al. (2010) Trends Neurosci. 33, 317-325;Sigurdsson, E. M. et al. (2002) Trends Mol. Med. 8, 411-413).

II. Tau Species

The discovery that the tau protein may act as an endo-pathogen hasspawned a search for “The Pathogenic Species” that could be targeted inpotential interventive therapies.

The microtubule-associated protein tau gene (MAPT) is located onchromosome 17 of the human genome and expresses six isoforms of the tauprotein in adult human brain. These isoforms arise from the alternativesplicing of exons 2, 3 and 10 of the 16 exons within the MAPT gene.Exons 2 and 3 express a 29 amino acid repeat and exon 10 expresses anadditional microtubule binding domain. As a result, tau isoforms willcontain 0, 1 or 2 N-terminal repeats and 3 or 4 C-terminal microtubulebinding domains (3R or 4R tau). Commonly six isoforms of tau areexpressed. The longest (2N4R) and shortest (0N3R) isoforms consist of441 and 352 amino acids, respectively (Kolarova, M. et al. (2012) Int.J. Alzheimers. Dis. 2012, 731526). The N-terminal projection domain oftau (2N4R) consists of a 44 amino acid glycine-rich tail and residues45-102 encompass two highly acidic regions (N1, N2-domains). Twoproline-rich regions are found at residues 151-243 (P1, P2 domains). Theremainder of the protein is constituted by four microtubule bindingdomains (R1-R4), followed by a short C-terminal region.

Tau is very soluble and highly phosphorylation-labile protein.Approximately 20 percent or 85 of the amino acid residues in the longestisoform of tau are potential (Ser, Thr or Tyr) phosphorylation sites.Roughly half of these have been observed experimentally (Hanger, D. P.et al. (2009) Trends Mol. Med. 15, 112-119; Hasegawa, M. et al. (1992)J. Biol. Chem. 267, 17047-17054), and are clustered around the terminalresidues of the microtubule binding domains. Tau is dynamicallyphosphorylated and de-phosphorylated during the cell cycle. It mustdissociate from microtubules to allow for meiosis to occur. Its mainrole in post mitotic cells (the differentiated neuron) is to act as amicrotubule stabilizer, allowing for optimal axonal transport. It canonly associate with microtubules in its mostly de-phosphorylated form,thus phosphorylation acts as a direct microtubuleassociation/dissociation switch within the neuron. Under normalconditions, cytosolic tau contains on average two phosphorylated sites.In paired helical filamentous material, at least 7-8 sites arephosphorylated (Hanger, D. P. et al. (2009) Trends Mol. Med. 15,112-119; Hasegawa, M. et al. (1992) J. Biol. Chem. 267, 17047-17054).Hyperphosphorylated, paired helical filamentous tau is a key hallmark ofAlzheimer's disease (Kosik et. al. (1986) PNAS, 86, 4044-4048), adistinct mobility shift of hyperphosphorylated tau is observed inimmune-cytochemical analysis of human AD brain material.

It has been difficult to study the tau protein with traditionalstructural techniques like x-ray crystallography or NMR spectroscopy,reflecting its meta-stable nature. Such studies have mainly beenconducted on domain fragments of the un-phosphorylated protein. The onlystructural study to date on full-length tau (2N4R), using NMRspectroscopy, reveals that the protein contains only sparse stretches ofstable secondary structure (Mukrasch, M. D. et al. (2009) PLoS. Biol. 7,e34). This analysis indicates that the secondary structure of thepeptide backbone has a large propensity for adapting a β-sheetstructure. The backbone's first 200 residues are considerably moreordered than the C-terminus encompassing the microtubule bindingdomains. The presence of many specific long-range interactions withinthe protein in solution indicates that it exists in a largely disorderedmolten globular state (Ohgushi, M. and Wada, A. (1983) FEBS Lett. 164,21-24).

Protease products of tau generated in particular by caspase and calpain(Asp13, Glu391 and Asp421) have been identified in tangle material(Gamblin, T. C. et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100,10032-10037). In particular, the truncation at Asp421 has been studiedin detail using the tau C3 antibody, which binds to the free Asp421terminus. This truncation has been postulated as an early event in ADpathogenesis associated with induction of apoptosis (deCalignon A. etal. (2010) Nature 464, 1201-1204). The N-terminal cleavage at Aspi3 andthe C-terminal cleavage at Glu391 are considered late events in thepathogenesis (deCalignon A. et al. (2010) Nature 464, 1201-1204;Delobel, P. et al. (2008) Am. J. Pathol. 172, 123-131). Recently, anadditional N-terminal fragment (residues 1-224) was identified in CSFfrom AD and PSP patients, and has been hypothesized to be an earlymarker of disease and particularly pathogenic (U.S. Ser. No. 14/092,539;Bright, J. et al. (2014) Neurobiol. Ageing, 1-17). A similar calpaincleaved fragment was reported by other groups (Ferreira, A. and Bigio,E. H. (2011) Mol. Med. 17, 676-685; Reinecke, J. B. et al. (2011) PLoS.One. 6, e23865).

Apart from hyper-phosphorylation and tau fragmentation,post-translational acetylation (Cohen, T. J. et al. (2011) Nat. Commun.2, 252; Min, S. W. et al. (2010) Neuron 67, 953-966) and O-GlcNAcylation(Zhu, Y. et al. (2014) J. Biol. Chem.) have been proposed to bepathology defining processes in the formation of tangle pathologyassociated with AD.

III. Tau Immunotherapies

Immunotherapies are traditionally separated into passive and activevaccine approaches. In an active vaccine approach, a pathogenic agent isinjected into the patient and the innate immune system elicits an immuneresponse. This triggers the maturation of B-cells generating highaffinity antibodies against the administered antigen. In a passivevaccine approach, the triggering of the innate immune system iscircumvented by infusing a specific antibody against the antigen. Theinherent clearance system then removes antibody bound ligand.

AC Immune is pursuing a mouse monoclonal antibody against phospho-serine409 of tau. Antibodies were profiled against human AD and control braintissue and were selected based on their ability to recognize tanglepathology. The humanized version of two antibodies, hACI-36-2B6-Ab1 andhACI-36-3A8-Ab1, both bind to a tau epitope within amino acids 401-418(WO 2013/151762).

The group of Roger Nitsch have isolated tau auto-antibodies from elderlyhealthy individuals with no sign of degenerative tauopathy. A number ofantibodies have been isolated using full length recombinant human tau(2N4R) to find tau specific antibodies. These were then screened fortheir ability to discriminate tau isolates from diseases and healthyindividuals. Three lead antibodies, 4E4, 4A3 and 24B2, have beendescribed in the patent literature (WO2012049570; US2012087861). Theirepitope mapping indicates that all recognize amino acids within andC-terminal to the microtubule binding region, from position V339 toK369. These antibodies do not exhibit any phospho-specificity.

C2N Diagnostics focus mainly on developing diagnostic tools for earlydetection of neurodegenerative disease. Antibodies were generatedagainst full length human and mouse tau protein. Eight and fiveantibodies were identified, recognizing human and mouse tau,respectively (Yanamandra, K. et al. (2013) Neuron 80, 402-414). Threeantibodies with different binding kinetics were selected for in vivoevaluation. Namely, HJ9.3, HJ9.4 and HJ8.5, recognizing tau residues306-320, 7-13 and 25-30, respectively, with the last one being specificfor human tau. The antibodies were also selected based on their abilityto prevent transfer of pathology in an ingenious mechanistic reporterassay of trans-cellular propagation of tau (Sanders, D. W. et al. (2014)Neuron 82, 1271-1288; Kfoury, N. et al. (2012) J. Biol. Chem. 287,19440-19451). Their evaluation in chronic i.c.v. injection studies inP301S transgenic mice demonstrated their ability to reduce levels ofhyper-phosphorylated tau protein as determined by AT8 staining inimmuno-histochemical analysis of the treated mice.

The antibodies of Peter Davies were developed originally as diagnostictools that could differentiate between pathological and normal tau in ADand control brain material (Greenberg, S. G. and Davies, P. (1990) Proc.Natl. Acad. Sci. U.S.A. 87, 5827-5831). Evaluation of the therapeuticutility of the PHF1 and MC1 antibodies was demonstrated in P301S andJPNL3 (P301L) (Boutajangout, A. et al. (2011) J. Neurochem. 118,658-667; Chai, X. et al. (2011) J. Biol. Chem. 286, 34457-34467;D'Abramo, C. et al. (2013) PLoS. One. 8, e62402 mice). PHF1 recognizes alinear phospho-tau epitope (pS396, pS404) whereas MC1 is aconformation-dependent antibody that recognizes a structural tau epitoperequiring two distinct parts of the linear sequence, an epitope withinresidues 46-202 and a C-terminal epitope between residues 312-342(Jicha, G. A. et al. (1997) J. Neurosci. Res. 48, 128-132). Injection ofthese two antibodies in chronic 12-13 week immunization studies resultedin substantial reduction of spinal cord and brainstem pathology amongother brain regions, which translated to an attenuation of the motordeficit observed in these mice. (D'Abramo, C. et al. (2013) PLoS. One.8, e62402).

iPerian/Bristol Meyers Squibb has developed tau antibodies against apostulated pathological tau species, composed of an N-terminal fragmentof tau (etau: residues 1-224), which promoted hyperactivity in inducedpluripotent stem cell based neuronal cultures. A portfolio of antibodieshas been developed, but characterization has focused on antibodiesIPN001 and IPN002 that recognize an N-terminal epitope within residues9-18. Accordingly, these antibodies detect elevated tau levels in CSFfrom staged AD and PSP patients that may be an early sign of disease. Invivo injections of the antibodies in JPNL3 (P301L) mice led to partialreversal of progressive motor deficits (U.S. Ser. No. 14/092,539).

Einar Sigurdsson were the first program to demonstrate the efficacy oftau based immunotherapy. An active vaccine consisting of tau peptide379-408[pS396, pS404] together with Adju-Phos adjuvant was used toimmunize JPNL3 (P301L) mice. In this study a prominent reduction of taupathology was observed in the vaccine treated mice when compared tocontrol animals. An attenuation of tauopathy-related motor phenotype wasdetected as well. Its efficacy was confirmed in a different mouse model(htau/PS1) not driven by mutant tau (Boutajangout, A. et al. (2011) AAIC2011 (7, issue 4, Supplement edn) p. s480-s431; Congdon, E. E. et al.(2013) J. Biol. Chem. 288, 35452-35465; Gu, J. et al. (2013) J. Biol.Chem. 288, 33081-33095).

Prothena has evaluated three tau antibodies in the K369 (K3) transgenictau mouse and in a P301L mouse model. Antibodies with varying propertieswere selected for in-vivo evaluation. Two pS404 antibodies withdifferent isotype (IgG1/k and IgG2a/k) or a total (pan) anti-tauantibody (IgG1/k) were injected in a chronic paradigm. K3691 mice weretreated with weekly injections for 21 weeks starting at 3 weeks of age,and P301L mice were treated for 7 months with weekly injections startingat 4 months of age. A reduction in tau positive neurofibrillaryinclusions was observed in the K3 mice with the IgG2a/k pS404 antibody.Both of the pS404 antibodies were able to reduce the levels of pS422positive tau, whereas no reduction was observed in the pan-anti-tauantibody treated mice. These studies suggest that: 1) tau clearance maybe isotype-dependent, and; 2) It may be important to target a tauspecies that is relevant to disease, as the total-anti-tau antibody wasunable to reduce hyper-phosphorylated tau (PCT/US2014/025044).

The inventors of the present invention have surprisingly foundantibodies specific for the phosphorylated tau serine residue 396(pS396); this is in contrast to the prior art antibodies which recognizeprimarily the tau proteins phosphorylated at both 396 and 404 residues,phosphorylated at the 404 residue only or at other residues on tau.

The inventors have developed antibodies which furthermore have aremarkable specificity and selectivity towards human pathological tau.There is a need for antibodies which are highly selective and specificfor pathogenic tau protein. The antibodies of the present invention showa much higher degree of specificity and selectivity towards humanpathological tau over non-pathological tau compared to the antibodies ofWO2013/050567 (see FIG. 1 of WO2013/050567). The antibodies ofWO2012/045882 reported to have a specific binding, were elicited from 6to 9 residue amino acid sequences of Tau amino acids 393-401, 396-401,394-400 and 393-400. This contrasts from the antibodies of the presentinvention which were elicited against pathogenic hyperphosphorylated taucomprising a longer amino acid sequence as described herein.

As shown in the Examples, comparison to five published tau antibodies:hACI-2B6 (described WO2013151762); IPN002 (described in WO 2014028777);HJ8.5 (described in WO 2014008404); the anti-Tau pS422 monoclonalantibody 2.10.3 (described in U.S. Pat. No. 8,609,097); PHF13 (acommercially available antibody (e.g. SigmaAldrich) recommended fordetection of Tau phosphorylated at Ser 396 of mouse, rat and humanorigin and discussed by Sankaranarayanan (PLOSONE,DOI:10.1371/journal.pone.0125614 May 1, 2015 and Otvos (Biochemistry1997, 36, 8114-8124); and the 4E4 antibody, (described as binding toV339, E342, D387, E391 and K395 in U.S. Pat. No. 8,940,272), showed thatthe antibodies, and epitope-binding fragments thereof, of the presentinvention exhibit a higher degree of specificity and selectivity towardshuman pathological tau than any of the comparator antibodies.

Further, the antibodies, and epitope-binding fragments thereof, of thepresent invention show many advantageous features such as the ability todiscriminate between pathological and non-pathological human tauprotein, and in particular to bind tau associated with Alzheimer's (AD)pathology. In electrophysiological studies, the antibodies, andepitope-binding fragments thereof, of the invention were additionallyable to reverse reduced paired pulse facilitation and spontaneousminiature excitatory synaptic current (mEPSC).

SUMMARY OF THE INVENTION

The present invention relates to monoclonal antibodies, andepitope-binding fragments thereof, capable of specifically binding tothe phosphorylated residue serine 396 of human (2N4R isoform) tau (SEQID NO:33) and to such antibodies that have been produced using a newmethod that allows such specific isolation and recovery. The antibodiesare further characterized by their ability to discriminate betweenphosphorylated residues 396 and 404 such that they substantially do notbind the phosphorylated 404 residue.

Without being bound by a particular theory, evidence from the inventorsdemonstrates that the discrimination and selectivity of the antibodiesof the present invention for human tau protein phosphorylated at residue396 in the presence of tau protein phosphorylated at residue 404 but notat 396 is significant from a pathological and therapeutic perspective.The antibodies of the present invention are selective for pathologicaltau in the presence of non-pathological—yet phosphorylated—tau. Theantibodies of the present invention are able to deplete tau tangles ofpathological tau in the presence of normal tau. Without being bound to aparticular theory, it is believed that depleting tangles of taucomprising tau protein that has been phosphorylated at tau position 396prevents seeding of pathological tau into tau tangles. Accordingly, oneaspect of the invention relates to an antibody that is capable ofselectively binding to 396-phosphorylated tau even when such moleculesare in the presence of tau protein that has been phosphorylated at tauposition 404. A related aspect of the invention relates to an antibodythat is capable of selectively binding to 396-phosphorylated tau evenwhen such molecules are in the presence of non-pathogenic tau. Furtherdefined, the invention relates to an antibody selective for pathologicaltau said pathological tau being hyperphosphorylated tau appearing as 64kDa band (by Western Blot analysis) in transgenic mice overexpressingthe human 2N4R isoform of tau.

One aspect of the invention is directed to an anti-tau antibody meetingthe following test criteria: i) the antibody does not bind tonon-phosphorylated tau; ii) the antibody does not bind to tauphosphorylated at 404 and not phosphorylated at 396; iii) the antibodydoes bind to tau phosphorylated at 396; and iv) the antibody does bindto tau phosphorylated at both 396 and 404. The inventors have found thatthe binding under test criteria iii) and iv) are in the same order ofmagnitude and postulate that phosphorylation at position 404 does notinterfere nor enhance the binding process. The inventors have furtherfound that, contrarily to test criteria ii), binding to a tau proteinwhich is not phosphorylated at 396 but is phosphorylated at 404, doesnot deplete tangles or clear pathological tau in test models.

One aspect of the invention is directed to an anti-tau antibody that,when used with immune-depleted rTg4510 extracts from transgenic mice,specifically reduces the hyperphosphorylated tau 64 and 70 kDa bands byat least 90%, while reducing the 55 kDa tau band by not more than 10%. Afurther aspect of the invention is directed to an anti-tau antibody thatspecifically reduces the hyperphosphorylated tau 64 and 70 kDa bands byat least 90%, while reducing the 55 kDa tau band by not more than 10%;or the capability, when used as described herein with extracts fromhuman AD post-mortem brains, to specifically reduce the phosphorylatedS396 hyperphosphorylated tau bands by at least 90%, while not reducingthe non-hyperphosphorylated tau bands by more than 10%.

Another aspect of the invention is directed to a method of treating apatient with a taupathy, such as Alzheimer's Disease, comprisingdepleting a tangle or attenuating the progression of said tangle, saidtangle comprising hyperphosphorylated Tau, said method comprisingcontacting hyperphosphorylated Tau with an antibody of the inventionsuch that the tangle is depleted, reduced in its content ofhyperphosphorylated tau or progression of tangle formation isattenuated.

Alternatively defined, the invention relates to a method of treating apatient with a taupathy, such as Alzheimer's Disease, said methodcomprising contacting tangles with an antibody selective for tau havingresidue 396 phosphorylated such that the tangle is depleted ofhyperphosphorylated Tau.

More specifically the invention relates to any one of four monoclonalantibodies selected from the group comprising:

Antibody C5.2

wherein Antibody C5.2 comprises:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:17;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:18;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:19;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:20;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:21;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:22;

Antibody C8.3

wherein Antibody C8.3 comprises:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:25;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:26;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:27;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:28;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:29;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:30;

Antibody C10-2

wherein Antibody C10-2 comprises:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14;

and

Antibody D1.2

wherein Antibody D1.2 comprises:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:1;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:2;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:3;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:4;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:5;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:6.

The amino acid sequences of the full light and heavy chains of anexemplary antibody C5.2, including the constant domains therein, areshown in SEQ ID NO:23 and SEQ ID NO:24, respectively (as used in theExamples).

The amino acid sequences of the full light and heavy chains of anexemplary antibody C8.3, including the constant domains therein, areshown in SEQ ID NO:31 and SEQ ID NO:32, respectively (as used in theExamples).

The amino acid sequences of the full light and heavy chains of aninteresting antibody C10-2, including the constant domains therein, areshown in SEQ ID NO:15 and SEQ ID NO:16, respectively (as used in theExamples). The amino acid sequence of the heavy chain of humanized C10-2antibody is shown in SEQ ID NO:35. The amino acid sequence of the lightchain of humanized C10-2 antibody is shown in SEQ ID NO:36. One aspectof the invention relates to an antibody of the invention comprising SEQID NO:35 or SEQ ID NO:36, or both.

The amino acid sequences of the full light and heavy chains of anexemplary antibody D1.2, including the constant domains therein, areshown in SEQ ID NO:7 and SEQ ID NO:8, respectively (as used in theExamples).

In an alternative embodiment, the antibody D1.2 comprises alight chainhaving the amino acid sequence of SEQ ID NO:34, wherein the amino acidat position 3 is valine (whereas in the exemplary light chain of SEQ IDNO:7, this amino acid is a methionine). This light chain may be pairedwith a heavy chain as described above, i.e. having CDRs of SEQ ID NOs:4,5 and 6. For example, the antibody may comprise a light chain having theamino acid sequence of SEQ ID NO:34 together with a heavy chain havingthe amino acid sequence of SEQ ID NO:8 (antibody “D1.2*”).

One aspect of the invention is directed to an antibody comprising:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10;and/or

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11.

A further aspect of the invention is directed to an antibody comprising,or additionally comprising:

(a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13;and/or

(c) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14.

The antibodies, and epitope-binding fragments thereof, of the inventioncan be used in treating tauopathies such as Alzheimer's disease (AD),Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP),Corticobasal Degeneration (CBD), TBI (traumatic brain injury, mild,acute or chronic), and chronic traumatic encephalopathy (CTE).

The antibodies, and epitope-binding fragments thereof, of the inventionare furthermore intended for use in treating Psychosis, particularlyPsychosis due to AD or Psychosis in patients with AD.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B: Binding to pathological material dot-blot

FIGS. 1A-1B present the results of a dot blot analysis displaying 500 ngS1 and P3 fractions (generation of the S1 and P3 fractions are disclosedin Example 3) derived from brains of AD patients (AD) and aged healthyindividuals (con) or from 32 weeks old rTg4510 and non-transgenic (wt)littermates probed with 1 μg/ml D1.2 or C10-2 to assess detection ofpathological tau (Example 3). The dot plot shows that D1.2 (FIG. 1A) orC10-2 (FIG. 1B) specifically reacts on disease material from AD patientsor human (P301L) tau as expressed in transgenic mice (Tg4510).

FIGS. 2A-2B: Western blot analysis of D1.2 and C10-2 antibodies

FIGS. 2A-2B present the results of a Western blot analysis displaying 2μg S1 and P3 fractions derived from brains of 32 week old rTg4510 andnon-transgenic (wt) littermates or 20 μg S1 and P3 fractions derivedfrom brains from AD patients (AD) and aged healthy individuals (con)probed with 1 μg/ml D1.2 (FIG. 2A) or C10-2 (FIG. 2B). S1 and P3fractions were loaded at a ratio of 1:50 (based on tissue weight) whichwas derived from 0.01 mg tissue. In Western blot, normal P301L mutanthuman 4R0N tau is displayed at 55 kDa, while hyper-phosphorylated P301Lmutant human 4R0N tau species is displayed at 64 and 70 kDa. In P3fractions from AD hyper-phosphorylated tau is displayed at 54, 64, 69and 74 kDa (Example 3). The figure illustrates that the antibodiesspecifically bind to hyper-phosphorylated, mobility shifted tau protein.

FIGS. 3A-3D: Binding to pathological P3 material in MSD

FIGS. 3A-3D present the results of Meso Scale Discovery (MSD) ELISAbinding of D1.2 (FIG. 3A), C5-2 (FIG. 3B), C10-2 (FIG. 3C) and C8-3(FIG. 3D) to tau isolated from human AD and non-diseased control brains(Example 4). Similar to what is demonstrated in FIGS. 1A-1B,immobilisation of tau isolated from disease (AD) and healthy controlbrains on ELISA plates can be used to demonstrate that the antibodies inthis invention specifically bind pathological tau species. Increasingconcentrations of antibody lead to saturation binding. The quantity ofbound antibody is detected with secondary anti-mouse antibody.

FIGS. 4A-4D: Peptide affinity and pS396 selectivity (peptide binding)

FIGS. 4A-4D present the results of an analysis of the specific bindingof C10-2 (FIG. 4A) and D1.2 (FIG. 4B) to tau (386-409) peptides with allcombinations of phosphorylation at positions S396 and S404 (Example 5).Specific affinity towards human pathological material is difficult toassess, for this reason we use specific peptide binding to determine theexact epitope affinity, using specific phosphorylated andun-phosphorylated peptides. Specific, dose response curves are shown forbinding of antibodies C10-2 (FIG. 4C) and D1.2 (FIG. 4D) to the peptide:TDHGAEIVYK^((p))SPVVSGDT^((p))SPRHL (SEQ ID NO:37) (pS396/pS404),phosphorylated at residues Ser396 and Ser404. Competition binding wasconducted with un-phosphorylated peptide (NP) and mono-phosphorylatedpeptides (pS396 and pS404). Additionally, a control peptidecorresponding to phosphorylated serine 262 was included. The competitionbinding demonstrates that all binding is obtained through thephosphorylated 396 serine residue. Additionally, the data demonstratesthat phosphorylation at residue 404 does not interfere with the bindingof antibodies at phosphor-serine 396.

FIGS. 5A-5B: Histological characterisation of pathology specificantibodies

FIG. 5A shows that C10-2 (left column) and D1.2 (right column)antibodies bind to p-tau species in Tg4510 (top row) cell bodies andneuropil. No immunoreactivity is detected in non-Tg brain sections(bottom row). FIG. 5B shows that C10-2 (left column) and D1.2 (rightcolumn) antibodies bind to p-tau species in cell bodies and neuropilthreads in AD donor (AD) (top row). Control donor brains are devoid ofimmunoreactivity (bottom row) (Example 6).

FIGS. 6A-6E: Binding to pathological and non-pathological P3 for C10-2and reference antibodies

FIGS. 6A-6E present results demonstrating the superiority of theantibody C10-2 (FIG. 6C) of the present invention in recognizingpathological material compared to prior art antibodies 2-10-3 (FIG. 6A),HACI-2B6 (FIG. 6B), IPN 002 (FIG. 6D), and HJ8.5 (FIG. 6E). The Figureshows the specific binding of C10-2 to tau from healthy (as a control)and disease (AD) human brains, together with the binding to tau from 10month old Tg4510 mice expressing P301L mutant human tau. Increasingconcentrations of antibody are added to P3 tau material immobilized onELISA plates. Ratio of selectivity towards pathological tau isdetermined at full saturation with active species. The fold selectivityfor each of the prior art antibodies is shown in the figure (Example 7).

FIGS. 7A-7C: Prevention of seeding in HEK293 cells and in-vitro

FIGS. 7A-7C present a quantification of tau aggregation by Cisbio assay.Seeded pcDNA HEK293 cells showed no signal, confirming the absence ofdetection for input seeding material. Wt (wild type) seeding material(WW) showed no seeding, but in contrast rTg4510 homogenates (CC) seededefficiently, compared to unseeded. This seeding effect was not affectedby treatment with HEL, but was partially reversed by treatment with tauantibodies (C10-2>D1.2>hACI36-2B6-Ab1). Graphs (FIGS. 7A-7C) representthree independent sets of samples and are plotted as relative tauaggregation (fold signal over background normalized to total protein)(Example 8).

FIGS. 8A-8D: Reversal of electrophysiological deficit

FIGS. 8A-8D show antibody reversal of paired pulse facilitation (FIG. 8Band FIG. 8D) and basal synaptic transmission (FIG. 8A and FIG. 8C)deficits in CA1 evoked field potentials (C10-2, FIG. 8A; D1.2, FIG. 8B),illustrating the evoked filed potentials in CA1 subchronic treatmentwith C10-2 in Tg4510 mice with and tTa mice as a control. Animals weretreated twice-weekly with a 15 mg/kg dose of antibody for two weeks (seeExample 9). In FIG. 8A (for C10-2) and FIG. 8C (for D1.2), the Fieldpotentials (fEPSP) slope is plotted against stimulation intensity. FIG.8A and FIG. 8C illustrate that in in vivo electrophysiologicalassessment of synaptic transmission and plasticity in the CA1 area ofthe hippocampus in 4.5 to 5.5 months old rTg4510 (lower 2 curves) andtTA (upper 2 curves) control mice i) basal synaptic transmission issignificantly impaired in rTg4510 compared to tTA mice, and ii)paired-pulse facilitation is significantly reduced rTg4510 compared totTA mice.

Paired-pulse facilitation, a short-term synaptic plasticity believed torely on presynaptic mechanisms, was further measured in rTg4510 and tTAmice (Panel B for C10-2 and Panel D for D1.2). Briefly, a pair ofstimuli with an inter-stimulus interval (ISI) varying from 25 to 1000 mswas applied to the Schaffer collateral, and the slope of the secondfEPSP was compared to the slope of the first fEPSP. Facilitation wasobserved at all ISIs, with a maximum facilitation at ISIs of 50 and 75ms. Interestingly, a significantly lower PPF was observed in rTg4510mice (second 2 bars) when compared tTA mice (first 2 bars).

FIG. 9: Overview of screening as outlined in FIGS. 1A-8D

Antibodies were raised against the bi-phosphorylated peptide:TDHGAEIVYK^((p))SPVVSGDT^((p))SPRHL (SEQ ID NO:37) covering residues386-410 of 2N4R tau. Hybridomas are screened using dot-blot and MSDELISA with immobilized human pathological and non-pathological tau(Example 4) to isolate clones that were highly specific towards theeither of the phospho-epitopes S396 and/or S404 and at the same timespecifically recognize hyper-phosphorylated tau from human Alzheimer'sdisease brains. The ability to discriminate between pathological andnon-pathological human tau protein in dot-blot and Western blot is usedfor selection of hybridomas. 16 clones were selected of which fourclones (D1.2, C10-2, C5.2 and C8.3) exhibit extraordinary capabilitiesfor binding to human pathological material. Use of the specificimmunization and screening protocol produces highly phospho-serine-396(pS396) specific antibodies.

FIG. 10: Residue pSer396 is bound at the center of the antigen bindingsite of mAb C5.2

The crystal structure of mAb C5.2 in a complex with phospho-peptide386-410 at 1.9 Å resolution. In this structure the electron density ofresidues 392-398 are resolved. Residue ^({p})Ser396 is bound at thecenter of the antigen binding site of mAb C5.2 In this structural studyof anti-Tau mAbs, the epitope is bound across the heavy chain (bottomright) and light (bottom left) chains.

FIG. 11: Antibody C5.2 interaction with phosphoserine tau (292-298)peptide

FIG. 11 represents the interaction between antibody C5.2 withphosphoserine tau (292-298) peptide. The structure ofIle(392)-VAL(393)-Tyr(394)-Lys(395)-P-Ser(396)-Pro(397)-Val(398) isshown. The main interaction involves the hydrophobic pocket formed byL3:H3, L3:F8*, H1:H13, H2:Y1, H2:Y3 and Y(394) of tau peptide. There isan extensive hydrogen bonding network formed between solvated^((p))S(396) and L3:T4, H1:R10, H1:T11, H3:R1, H3:T3. In the employednomenclature, the first letter (e.g., “L” of L3:H3) denotes whether theinvolved CDR residue is alight chain CDR or a heavy chain CDR, the firstnumber denotes which CDR of such chain is involved (e.g., “L3” denotesCDR3 of the light chain), the remaining terms (e.g., “H3” of L3:H3)denote the name and position of the involved amino acid (e.g., “H3”denotes a histidine at the third residue position of the CDR); thus“L3:H3” denotes the histidine residue at the third position of the oflight chain CDR3. There are strong hydrogen bonding and charge/polarinteractions between the Y(394) sidechain and the backbone withphosphonate of ^((p))S396 forms turn in peptide backbone. (*) L3:F8 isthe C-terminal flanking framework residue of CDR L3.

The CDR sequences of C5.2 are:

CDR L1: (SEQ ID NO: 17) QASQDTSINLN CDR L2: (SEQ ID NO: 18) GASNLEDCDR L3: (SEQ ID NO: 19) LQHTYLP CDR H1: (SEQ ID NO: 20) KASGYTFTDRTIHCDR H2: (SEQ ID NO: 21) YIYPGDDSTKYNDNFKG CDR H3: (SEQ ID NO: 22) RGTMDY

FIGS. 12A-12B: Depletion of Tau for seeding assay (HEK293)

FIGS. 12A-12B show immuno-depletion of rTg4510 brain homogenates usingmurine C10-2 (mC10-2) and humanized C10-2 (hC10-2). Western blots ofdepleted homogenates were detected with E1 (total tau; FIG. 12A; Lower))and C10-2 (pS396 tau; FIG. 12A; Upper) and both mC10-2 and hC10-2efficiently depleted hyperphosphorylated tau (upper bands on E1 blot andall bands on C10-2 blot). Depleted homogenates were also analyzed forthe depletion of aggregated tau using the Cisbio assay. FIG. 12B showsthe change in aggregated Tau in samples. Depletion studies with mC10-2and hC10-2 removed tau aggregates by 99 and 99.5% respectively (FIG.12B).

FIGS. 13A-13C: Seeding assay (HEK293) with depleted material

FIGS. 13A-13C show depleted homogenates used to seed P301L-hTau inHEK293 cells. Homogenates from control animals (WW) did not seed,whereas rTg4510 homogenates (CC) seeded efficiently, as measured by theCisbio aggregation assay on total cell lysates or by fractionation ofHEK293 cells in 1% triton-X (quantification of insolublehyperphosphorylated D1.2 and tau (FIG. 13A, Upper and Lower)). Depletionwith HEL and hHEL antibodies did not affect seeding, whereas depletionwith mC10-2 and hC10-2 prevented tau aggregation 88% and 96% (FIG. 13C)and insoluble tau 97% and 100% (FIG. 13B) respectively.

FIG. 14A-14C: Immuno depletion rTg4510 material (used for in vivoseeding studies)

FIGS. 14A-14C demonstrate Western blot (FIG. 14A; Upper, Lower) analysisof immuno-depleted rTg4510 brain extracts. C10-2 and D1.2 specificallyreduce the human hyperphosphorylated 64 kDa band by 90% and has noeffect on the 55 kDa Tau Tau5, a commercial total Tau antibody does incontrast reduce normal 55 kDa Tau by 74% and no effect on the human 64kDa Tau (FIGS. 14B-14C).

FIGS. 15A-15C: Immuno depletion AD material (used for in vivo seedingstudies)

FIGS. 15A-15C depict Western blot (FIG. 15A) analysis of immuno-depletedAlzheimer brain extracts. Immuno-depletion using C10-2 and D1.2 does notreduce the total Tau levels by more than 10%, but specifically lowerhyperphosphorylated Tau (90% reduction) (FIGS. 15B-15C).

FIG. 16A: Hippocampal Tau pathology in rTg4510 mice seeded with immunodepleted rTg4510 material

FIG. 16A illustrates the quantification of Tau pathology in rTg4510brains seeded with rTg4510 or AD brain homogenates. Prior to seeding thehyperphosphorylated Tau, but not normal Tau, had been reduced in thehomogenates by 90-95% by using C10-2 or D1.2. By removinghyperphosphorylated tau from the homogenates, the homogenates do nolonger induce seeding of Tau pathology.

FIG. 16B: Hippocampal tangle pathology in rTg4510 mice seeded withimmuno depleted AD material

FIG. 16B illustrates the quantification of Tau pathology in rTg4510brains seeded with rTg4510 (A) or AD (B) brain homogenates. Prior toseeding the hyperphosphorylated Tau, but not normal Tau, had beenreduced in the homogenates by 90-95% by using C10-2 or D1.2. By removinghyperphosphorylated tau from the homogenates, the homogenates do nolonger induce seeding of Tau pathology.

FIG. 17: Hippocampal tangle pathology in seeded rTg4510 mice treatedwith D1.2

FIG. 17 depicts the quantification of tangle bearing neurons inhippocampus of seeded rTg4510 mice. The pathology increases with time(Ig G, 1 month; IgG 2 months; IgG 3 months). However, treating the micewith D1.2, the pathology is significantly lowered 1, 2 and 3 monthsafter seeding. (D1.2 1 month; D1.2 2 months; D1.2 3 months).

FIGS. 18A-18B: Depletion of Tau for seeding assay (HEK293)

FIG. 18A shows immuno-depletion of rTg4510 brain homogenates usingmurine C10-2 (mC10-2) and humanized C10-2 (hC10-2). Western blots ofdepleted homogenates were detected with E1 (total tau) and C10-2 (pS396tau) and both mC10-2 and hC10-2 efficiently depleted hyperphosphorylatedtau (upper bands on E1 blot and all bands on C10-2 blot). Depletedhomogenates were analyzed for the depletion of aggregated tau using theCisbio assay. FIG. 18B shows depletion with mC10-2 and hC10-2 removedtau aggregates 99 and 99.5% respectively.

FIGS. 19A-19C: Seeding assay (HEK293) with depleted material

FIG. 19A shows Tau fractionation (western on insoluble fraction. FIG.19B shows Western blow quantification. FIG. 19C shows aggregated Tau incell lysates. Depleted homogenates were used to seed P301L-hTau inHEK293 cells. Homogenates from control animals (WW) did not seed,whereas rTg4510 homogenates (CC) seeded efficiently, as measured by theCisbio aggregation assay on total cell lysates or by fractionation ofHEK293 cells in 1% triton-X (quantification of insolublehyperphosphorylated D1.2+ tau). Depletion with HEL and hHEL antibodiesdid not affect seeding, whereas depletion with mC10-2 and hC10-2 (FIG.19C) prevented tau aggregation by 88% and 96% and insoluble tau by 97%and 100% respectively (FIG. 19B).

FIG. 20A-20B: Immunoselectivity of C10-2 and D1.2 forhyperphopsphorylated tau over normal tau.

Immuno depletion rTg4510 material used for in vivo seeding studies: FIG.20A shows Western blot analysis of immuno-depleted rTg4510 brainextracts. Panel B shows that C10-2 and D1.2 specifically reduce thehyperphosphorylated 64 kDa band, phosphorylated at serine 396 over theTau 55 kDa band, which does not comprise a significant amount of p396.In contrast, Tau5, a commercial total Tau antibody, does reduce normal55 kDa Tau and is inefficient in binding to the 64 kDa Tau.

FIGS. 21A-21C: Immunoselectivity of C10-2 and D1.2 forhyperphopsphorylated tau over normal tau.

Immuno depletion AD material (used for in vivo seeding studies: FIG. 21Ashows Western blot analysis of immuno-depleted Alzheimer brain extracts.Immuno-depletion using mC10-2 and D1.2 does not reduce the total Taulevels by more than 10% (FIG. 21B), but specifically lowerhyperphosphorylated Tau (90% reduction) (FIG. 21C).

FIGS. 22A-22B: Hippocampal Tau pathology in rTg4510 mice

FIG. 22A shows hippocampal Tau pathology in rTg4510 mice seeded withimmuno depleted rTg4510 material. FIG. 22B shows hippocampal tanglepathology in rTg4510 mice seeded with immuno depleted AD material.Quantification of Tau pathology in rTg4510 brains seeded with rTg4510(A) or AD (B) brain homogenates. Prior to seeding thehyperphosphorylated Tau, but not normal Tau, had been reduced in thehomogenates by 90-95% by using antibodies C10-2 or D1.2. By removinghyperphosphorylated tau from the homogenates, the homogenates no longerinduce seeding of Tau pathology.

FIG. 23: Hippocampal tangle pathology in seeded rTg4510 mice treatedwith D1.2

Quantification of tangle bearing neurons in hippocampus of seededrTg4510 mice. The Figure shows that pathology increases with time and bytreating the mice with D1.2, the pathology is significantly lower 2 and3 months after seeding.

FIG. 24: Western blot analysis of immuno-depleted human AD extracts

The Figure illustrates that humanized version of C10-2 (hC10-2), as wellas mC10-2 differ from the 2.10.3 (P-S422) antibody, in that althoughtotal tau remaining is not dramatically different (left hand panel) from2.10.3, C10-2 (hC10-2), as well as mC10-2 remove more of thehyperphosphorylated Tau protein present in Alzheimer brain extracts byimmuno depletion methods. This is confirmed in FIG. 25 byquantification.

FIG. 25: Quantification of Aggregated Tau after immuno depletion

The hC10-2 and mC10-2 antibodies differ from the 2.10.3 antibody in itsability to removes more of the aggregated Tau protein present inAlzheimer brain extracts by immuno depletion methods.

FIG. 26: Total tau remaining after immuno depletion

Quantification of western blot signal after immuno depleting Alzheimerextracts using different amounts of the humanized C10-2 (▴) and 2.10.3antibody (♦). In FIG. 26, quantification of total tau signal using Tau5(all tau isoforms were included in the analysis) is shown. Bothantibodies remove a small fraction of tau from the Alzheimer brainpreparation. 2.10.3, designed to have specificity for P-S422 tau removesup to 24% of the total tau amount, while C10-2 removes up to 15% of thetotal tau (see FIG. 26).

FIG. 27: Total tau remaining after immunodepletion ofhyperphosphorylated tau

FIG. 27 illustrates the quantification of the hyperphosphorylated tau,being phosphorylated at serine 422 (all bands and the high molecularweight smear was included in the analysis). 2.10.3 (▴) and C10-2 (♦)both remove more than 90% of the tau phosphorylated at Serine 422.However, the amount of antibody needed to remove 50% of the tau differ:for antibody 2.10.3, 0.42 μg antibody is needed whereas for C10-2, 0.27μg is needed for the same effect.

FIG. 28: Total tau remaining after immunodepletion ofhyperphosphorylated tau

Quantification of the hyperphosphorylated tau, being phosphorylated atserine 396 (all bands and the high molecular weight smear was includedin the analysis). C10-2 (♦) efficiently removes Tau being phosphorylatedat serine 396 (Max effect: 88% and half of the effect is reached byusing 0.30 μg antibody). 2.10.3 (▴) removes a smaller fraction of taubeing phosphorylated at the serine 396 (Max effect: 60% and half of thateffect is reached when using 0.63 μg antibody). This indicates that allTau being phosphorylated at serine 422, also is phosphorylated at serine396, but that there is a portion of hyperphosphorylated tau beingphosphorylated at serine 396 where the phosphorylated serine at position422 is not present.

FIG. 29: Total tau remaining after immunodepletion ofhyperphosphorylated tau

Quantification of the hyperphosphorylated tau, being phosphorylated atserine 199/202 (all bands and the high molecular weight smear wasincluded in the analysis). A large portion of the tau being removed byC10-2 (♦), is also phosphorylated at Serine 199/202, since 69% of thetau having that phosphporylation is affected by the immunodepletion (50%of the effect when using 0.34 μg antibody). The 2.10.3 (▴)immunodepletion does not give a sigmoidal dose response on theP-S199/202 tau although a drop in signal is seen with increasing amountof antibody (max 52% reduction when using the max amount of antibody (5μg). This data indicates that the C10-2 antibody targeting thephosphorylated serine 396 binds a larger pool of the hyperphosporylatedtau then the 2.10.3 antibody targeting the phosphorylated serine at the422 position.

FIG. 30: mD1.2 and mC10-2 inhibition of Tau antigen capture in mC10-2coated plates

In fluid phase ELISA, where a mixture of rTg4510 P3 preparation andvariable amounts of C10-2 or D1.2 antibodies is added onto C10-2 coatedplates. The more antibodies binding to P3 tau in the solution, lessavailable tau epitopes are able to bind to the plates. The amount of taubinding to the plates is determined by a sulfo-tagged human tauantibody. C10-2 (▴) and D1.2 (□) have a different binding to the tau insolution, wherein C10-2 can compete out all binding to the plates (IC5020 nM). D1.2, on the other hand, shows a very low level of binding tothe tau in the solution.

FIG. 31: PHF13 and mC10-2 inhibition of Tau antigen capture in mC10-2coated plates

In fluid phase ELISA, a mixture of AD P3 preparation and variableamounts of C10-2 and PHF13 antibodies were added onto C10-2 coatedplates. The more antibodies binding to P3 tau in the solution, the lessavailable tau epitopes are able to bind to the plates. The amount of taubinding to the plates is determined by a sulfo-tagged human tauantibody. C10-2 and PHF13 have a different binding to the tau insolution, wherein C10-2 can compete out all binding to the plates(IC50=3 nM), whereas PHF13 does not.

FIG. 32: Both mC10-2 and PFH-13 bind dose dependently to Ptau 386-408(pS396/pS404)

FIG. 32 shows mC10-2 and PHF-13 bind equally well in MSD plates coatedwith 100 ng/ml p-tau 386-408 (pS396/pS404. Increasing concentrations ofantibodies (indicated on x-axis) was incubated in wells for 2 hrsfollowed by wash and detection of bound antibodies using sulfo taggedanti-human IgG antibodies. This indicates that the prepared PHF-13 usedin subsequent examples is active.

FIG. 33: Comparing mD1.2 and mC10-2 binding to AD-P3

FIG. 33 shows mD1.2 and mC10-2 binds equally well in MSD plates coatedwith 1 μg/ml AD-P3. Increasing concentrations of antibodies (indicatedon x-axis) incubated in the presence and absence of 10 uM p-tau 386-408(pS396/pS404) peptide for 1 hour at room temp followed by incubation inwells for 2 hours followed prior to detection of bound antibodies usingsulfo tagged anti-human IgG antibodies. IC50 values were 320 nM and 11nM for capture of AD-P3 and AD-S1(p). In contrast mD1.2 showedsignificantly weaker inhibition of tau antigen capture with IC50 valuesof 589 and 503 nM—suggesting much lower affinity binding to solubleantigens.

Assay was performed in two steps A: 1 μg/ml AD-P3 and 20 ng/ml AD si(p),respectively was incubated with increasing concentration of mD1.2 andmC10-2 and incubated 1 hour at room temperature to allow increasingantibody-antigen binding (occupancy). B: The samples were incubated onMSD plates coated with AD-P3 (1 μg/ml) for 2 hours followed by wash anddetection of captured Tau antigens using sulfo tagged anti-total TauGantibodies.

FIG. 34: mC10-2 but not PHF-13 binds efficiently to solid phasedisplayed AD-P3 antigens Highly specific binding of mC10-2 but notPHF-13: FIG. 34 shows that mC10-2 bind efficiently to AD-P3 antigenscoated MSD plates (1 μg/ml). In comparison, the low binding activity ofPHF-13 indicates lower affinity to physiological p-tau Antigens.Furthermore PHF-13 demonstrated substantial higher degree ofnon-specific binding in comparison to mC10-2 (see Table 6). Increasingconcentrations of antibodies (indicated on x-axis) incubated for 2 hoursfollowed prior to detection of bound antibodies using sulfo taggedanti-human IgG antibodies. Binding signal was corrected for non-specificbinding activity (defined as signals measured in presence of 10 uM p-tau386-408 (pS396/pS404) peptide. IC50 value were 3 nM for mC10-2 captureof AD-P3. In contrast PHF-13 showed virtually no inhibition.

Assay was performed in two steps. A: 1 μg/ml AD-P3 was incubated withincreasing concentration of mC10-2 and PHF-13 and incubated 1 hour atroom temperature to allow increasing antibody-antigen binding(occupancy).B: The samples were incubated on MSD plates coated withAD-P3 (1 μg/ml) for 2 hours followed by wash and detection of capturedTau antigens using sulfo tagged anti-total Tau antibodies.

SEQUENCES INCORPORATED BY REFERENCE SEQ ID NO: 1 D1.2 Light Chain CDR1SEQ ID NO: 2 D1.2 Light Chain CDR2 SEQ ID NO: 3 D1.2 Light Chain CDR3SEQ ID NO: 4 D1.2 Heavy Chain CDR1 SEQ ID NO: 5 D1.2 Heavy Chain CDR2SEQ ID NO: 6 D1.2 Heavy Chain CDR3 SEQ ID NO: 7 D1.2 Light Chain SEQ IDNO: 8 D1.2 Heavy Chain SEQ ID NO: 9 C10-2 Light Chain CDR1 SEQ ID NO: 10C10-2 Light Chain CDR2 SEQ ID NO: 11 C10-2 Light Chain CDR3 SEQ ID NO:12 C10-2 Heavy Chain CDR1 SEQ ID NO: 13 C10-2 Heavy Chain CDR2 SEQ IDNO: 14 C10-2 Heavy Chain CDR3 SEQ ID NO: 15 C10-2 Light Chain SEQ ID NO:16 C10-2 Heavy Chain SEQ ID NO: 17 C5.2 Light Chain CDR1 SEQ ID NO: 18C5.2 Light Chain CDR2 SEQ ID NO: 19 C5.2 Light Chain CDR3 SEQ ID NO: 20C5.2 Heavy Chain CDR1 SEQ ID NO: 21 C5.2 Heavy Chain CDR2 SEQ ID NO: 22C5.2 Heavy Chain CDR3 SEQ ID NO: 23 C5.2 Light Chain SEQ ID NO: 24 C5.2Heavy Chain SEQ ID NO: 25 C8.3 Light Chain CDR1 SEQ ID NO: 26 C8.3 LightChain CDR2 SEQ ID NO: 27 C8.3 Light Chain CDR3 SEQ ID NO: 28 C8.3 HeavyChain CDR1 SEQ ID NO: 29 C8.3 Heavy Chain CDR2 SEQ ID NO: 30 C8.3 HeavyChain CDR3 SEQ ID NO: 31 C8.3 Light Chain SEQ ID NO: 32 C8.3 Heavy ChainSEQ ID NO: 33 Human tau SEQ ID NO: 34 D1.2* Light Chain SEQ ID NO: 35humanized C10-2 Heavy Chain SEQ ID NO: 36 humanized C10-2 Light ChainSEQ ID NO: 37 tau residues 386-408 (pS396, pS404)

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “tau” is synonymous with “the tau protein” andrefers to any of the tau protein isoforms (identified in, for example,UniProt as P10636, 1-9). The amino acid numbering of tau that is usedherein is given with respect to isoform 2 (SEQ ID NO:33) as shown below,with methionine (M) being amino acid residue 1:

SEQ ID NO: 33 MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTDAGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLVDEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAGHVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPPGQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSPGSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAKSRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINKKLDLSNVQSK CGSKDNIKHV PGGGSVQIVY KPVDLSKVTSKCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNITHVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVSGDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG L

The present invention relates to antibodies and epitope-bindingfragments thereof that are capable of specifically binding to tau, andin particular to human tau, and in one embodiment exhibit the ability tospecifically bind to the phosphorylated S396 residue (pS396) of humantau. The antibodies and epitope-binding fragments thereof of theinvention, are further characterized by being incapable or substantiallyincapable of specifically binding to the phosphorylated 404 (pS404)residue on human tau, for example under antibody limited ornon-saturating conditions. Furthermore, phosphorylation at pS404 doesnot interfere with the specific binding to pS396. As used herein, thenotations “pS” and “^((p))S” denote the amino acid residuephosphoserine. As used herein, an antibody is “substantially” incapableof binding to an epitope if relative to another epitope such binding isless than 20%, less than 10%, less than 5%, less than 2%, and morepreferably, less than 1% of the binding observed with such otherepitope.

The term “antibody” (Ab) in the context of the present invention refersto an immunoglobulin molecule or according to some embodiments of theinvention, a fragment of an immunoglobulin molecule which has theability to specifically bind to an epitope of a molecule (“antigen”).Naturally occurring antibodies typically comprise a tetramer which isusually composed of at least two heavy (H) chains and at least two light(L) chains. Each heavy chain is comprised of a heavy chain variabledomain (abbreviated herein as VH) and a heavy chain constant domain,usually comprised of three domains (CH1, CH2 and CH3). Heavy chains canbe of any isotype, including IgG (IgG1, IgG2, IgG3 and IgG4 subtypes).Each light chain is comprised of a light chain variable domain(abbreviated herein as VL) and a light chain constant domain (CL). Lightchains include kappa chains and lambda chains. The heavy and light chainvariable domain is typically responsible for antigen recognition, whilethe heavy and light chain constant domain may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (C1q)of the classical complement system. The VH and VL domains can be furthersubdivided into domains of hypervariability, termed “complementaritydetermining regions,” that are interspersed with domains of moreconserved sequence, termed “framework regions” (FR). Each VH and VL iscomposed of three CDR Domains and four FR Domains arranged fromamino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable domains of the heavy andlight chains contain a binding domain that interacts with an antigen. Ofparticular relevance are antibodies and their epitope-binding fragmentsthat have been “isolated” so as to exist in a physical milieu distinctfrom that in which it may occur in nature or that have been modified soas to differ from a naturally occurring antibody in amino acid sequence.

The term “epitope” means an antigenic determinant capable of specificbinding to an antibody. Epitopes usually consist of surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and linear epitopes aredistinguished in that the binding to the former, but not the latter, isalways lost in the presence of denaturing solvents. The epitope maycomprise amino acid residues directly involved in the binding and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically epitope-binding peptide (in other words, the amino acidresidue is within the footprint of the specifically epitope-bindingpeptide).

As used herein, the term “epitope-binding fragment of an antibody” meansa fragment, portion, region or domain of an antibody (regardless of howit is produced (e.g., via cleavage, recombinantly, synthetically, etc.))that is capable of specifically binding to an epitope. Anepitope-binding fragment may contain 1, 2, 3, 4, 5 or all 6 of the CDRDomains of such antibody and, although capable of specifically bindingto such epitope, may exhibit a specificity, affinity or selectivitytoward such epitope that differs from that of such antibody. Preferably,however, an epitope-binding fragment will contain all 6 of the CDRDomains of such antibody. An epitope-binding fragment of an antibody maybe part of, or comprise, a single polypeptide chain (e.g., an scFv), ormay be part of, or comprise, two or more polypeptide chains, each havingan amino-terminus and a carboxyl terminus (e.g., a diabody, a Fabfragment, a Fab₂ fragment, etc.). Fragments of antibodies that exhibitepitope-binding ability can be obtained, for example, by proteasecleavage of intact antibodies. More preferably, although the two domainsof the Fv fragment, VL and VH, are naturally encoded by separate genes,polynucleotides that encode such gene sequences (e.g., their encodingcDNA) can be joined, using recombinant methods, by a flexible linkerthat enables them to be made as a single protein chain in which the VLand VH regions associate to form monovalent epitope-binding molecules(known as single-chain Fv (scFv); see e.g., Bird et al., (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. (U.S.A.)85:5879-5883). Alternatively, by employing a flexible linker that is tooshort (e.g., less than about 9 residues) to enable the VL and VH domainsof a single polypeptide chain to associate together, one can form abispecific antibody, diabody, or similar molecule (in which two suchpolypeptide chains associate together to form a bivalent epitope-bindingmolecule) (see for instance PNAS USA 90(14), 6444-8 (1993) for adescription of diabodies). Examples of epitope-binding fragmentsencompassed within the present invention include (i) a Fab′ or Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains, or a monovalent antibody as described in WO2007059782; (ii)F(ab′)2 fragments, bivalent fragments comprising two Fab fragmentslinked by a disulfide bridge at the hinge domain; (iii) an Fd fragmentconsisting essentially of the VH and CH1 domains; (iv) a Fv fragmentconsisting essentially of a VL and VH domains, (v) a dAb fragment (Wardet al., Nature 341, 544-546 (1989)), which consists essentially of a VHdomain and also called domain antibodies (Holt et al; Trends Biotechnol.2003 November; 2i(II):484-90); (vi) camelid or nanobodies (Revets et al;Expert Opin Biol Ther. 2005 January; 5 (I): II-24) and (vii) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they may be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH domains pair to form monovalent molecules (known as single chainantibodies or single chain Fv (scFv), see for instance Bird et al.,Science 242, 423-426 (1988) and Huston et al., PNAS USA 85, 5879-5883(1988)). These and other useful antibody fragments in the context of thepresent invention are discussed further herein. It also should beunderstood that the term antibody, unless specified otherwise, alsoincludes antibody-like polypeptides, such as chimeric antibodies andhumanized antibodies, and antibody fragments retaining the ability tospecifically bind to the antigen (epitope-binding fragments) provided byany known technique, such as enzymatic cleavage, peptide synthesis, andrecombinant techniques. An antibody as generated can possess anyisotype. As used herein, “isotype” refers to the immunoglobulin class(for instance IgG1, IgG2, IgG3 or IgG4) that is encoded by heavy chainconstant domain genes. Such antibody fragments are obtained usingconventional techniques known to those of skill in the art; suitablefragments capable of binding to a desired epitope may be readilyscreened for utility in the same manner as an intact antibody.

The term “bispecific antibody” refers to an antibody containing twoindependent epitope-binding fragments that each target independenttargets. These targets can be epitopes present on different proteins ordifferent epitopes present on the same target. Bispecific antibodymolecules can be made using compensatory amino acid changes in theconstant domains of the HCs of the parent monospecific bivalent antibodymolecules. The resulting heterodimeric antibody contains one Fabscontributed from two different parent monospecific antibodies. Aminoacid changes in the Fc domain leads to increased stability of theheterodimeric antibody with bispecificity that is stable over time.(Ridgway et al., Protein Engineering 9, 617-621 (1996), Gunasekaran etal., JBC 285, 19637-1(2010), Moore et al., MAbs 3:6 546-557 (2011),Strop et al., JMB 420, 204-219 (2012), Metz et al., Protein Engineering25:10 571-580 (2012), Labrijn et al., PNAS 110:113, 5145-5150 (2013),Spreter Von Kreudenstein et al., MAbs 5:5 646-654 (2013)). Bispecificantibodies can also include molecules that are generated using ScFvfusions. Two monospecific scfv are then independently joined to Fcdomains able to form stable heterodimers to generate a single bispecificmolecule (Mabry et al., PEDS 23:3 115-127 (2010). Bispecific moleculeshave dual binding capabilities.

The term “antibody D1.2” is intended to denote an antibody or anepitope-binding fragment thereof, comprising, or consisting of, anantibody Light Chain Variable domain having:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:1;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:2;and

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:3;

and an antibody Heavy Chain Variable domain having:

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:4;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:5;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:6.

In one embodiment, the antibody D1.2 or epitope-binding fragment thereofmay comprise or consist of the heavy chain of SEQ ID NO:8 and/or thelight chain of SEQ ID NO:7.

In a related embodiment, the antibody D1.2* or epitope-binding fragmentthereof may comprise or consist of the heavy chain of SEQ ID NO:8 and/orthe light chain of SEQ ID NO:34.

The term “antibody C10-2” is intended to denote an antibody or anepitope-binding fragment thereof comprising, or consisting of, anantibody Light Chain Variable domain having:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10;and

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11;

and an antibody Heavy Chain Variable domain having:

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14.

In one embodiment, the antibody C10-2 or epitope-binding fragmentthereof may comprise or consist of the heavy chain of SEQ ID NO:16and/or the light chain of SEQ ID NO:15.

In a further embodiment, the humanized the antibody C10-2 orepitope-binding fragment thereof may comprise or consist of the heavychain of SEQ ID NO:35, the light chain of SEQ ID NO:36, or both. Oneembodiment of the invention is directed to an antibody orepitope-binding fragment thereof comprising or consisting of the heavychain of SEQ ID NO:35, the light chain of SEQ ID NO:36.

The term “antibody C5.2” is intended to denote an antibody orepitope-binding fragment thereof comprising, or consisting of, anantibody Light Chain Variable domain having:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:17;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:18;and

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:19;

and an antibody Heavy Chain Variable domain having:

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:20;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:21;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:22.

In one embodiment, the antibody C5.2 or epitope-binding fragment thereofmay comprise or consist of the heavy chain of SEQ ID NO 24 and/or thelight chain of SEQ ID NO:23.

The term “antibody C8.3” is intended to denote an antibody or anepitope-binding fragment thereof fragment thereof comprising, orconsisting of, an antibody Light Chain Variable domain having:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:25;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:26;and

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:27;

and an antibody Heavy Chain Variable domain having:

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:28;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:29;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:30.

In one embodiment, the antibody C8.3 or epitope-binding fragment thereofmay comprise or consist of the heavy chain of SEQ ID NO 32 and/or thelight chain of SEQ ID NO:31.

An “anti-tau antibody” is an antibody or an epitope-binding fragmentthereof, which binds specifically to tau or a tau fragment.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A conventional monoclonal antibody compositiondisplays a single binding specificity and affinity for a particularepitope. In certain embodiments a monoclonal antibody can be composed ofmore than one Fab domain thereby increasing the specificity to more thanone target. The terms “monoclonal antibody” or “monoclonal antibodycomposition” are not intended to be limited by any particular method ofproduction (e.g., recombinant, transgenic, hybridoma, etc.).

The antibodies of the present invention and epitope-binding fragmentsthereof will preferably be “humanized,” particularly if employed fortherapeutic purposes. The term “humanized” refer to a molecule,generally prepared using recombinant techniques, having anepitope-binding site derived from an immunoglobulin from a non-humanspecies and a remaining immunoglobulin structure based upon thestructure and/or sequence of a human immunoglobulin. The epitope-bindingsite may comprise either complete non-human antibody variable domainsfused to human constant domains, or only the complementarity determiningregions (CDRs) of such variable domains grafted to appropriate humanframework regions of human variable domains. The framework residues ofsuch humanized molecules may be wild type (e.g., fully human) or theymay be modified to contain one or more amino acid substitutions notfound in the human antibody whose sequence has served as the basis forhumanization. Humanization lessens or eliminates the likelihood that aconstant domain of the molecule will act as an immunogen in humanindividuals, but the possibility of an immune response to the foreignvariable domain remains (LoBuglio, A. F. et al. (1989) “Mouse/HumanChimeric Monoclonal Antibody In Man: Kinetics And Immune Response,”Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224). Another approach focusesnot only on providing human-derived constant domains, but modifying thevariable domains as well so as to reshape them as closely as possible tohuman form. It is known that the variable domains of both heavy andlight chains contain three complementarity-determining regions (CDRs)which vary in response to the antigens in question and determine bindingcapability, flanked by four framework regions (FRs) which are relativelyconserved in a given species and which putatively provide a scaffoldingfor the CDRs. When nonhuman antibodies are prepared with respect to aparticular antigen, the variable domains can be “reshaped” or“humanized” by grafting CDRs derived from nonhuman antibody on the FRspresent in the human antibody to be modified. Application of thisapproach to various antibodies has been reported by Sato, K. et al.(1993) Cancer Res 53:851-856. Riechmann, L. et al. (1988) “ReshapingHuman Antibodies for Therapy,” Nature 332:323-327; Verhoeyen, M. et al.(1988) “Reshaping Human Antibodies: Grafting An Antilysozyme Activity,”Science 239:1534-1536; Kettleborough, C. A. et al. (1991) “HumanizationOf A Mouse Monoclonal Antibody By CDR-Grafting: The Importance OfFramework Residues On Loop Conformation,” Protein Engineering4:773-3783; Maeda, H. et al. (1991) “Construction Of Reshaped HumanAntibodies With HIV-Neutralizing Activity,” Human Antibodies Hybridoma2:124-134; Gorman, S. D. et al. (1991) “Reshaping A Therapeutic CD4Antibody,” Proc. Natl. Acad. Sci. (U.S.A.) 88:4181-4185; Tempest, P. R.et al. (1991) “Reshaping A Human Monoclonal Antibody To Inhibit HumanRespiratory Syncytial Virus Infection in vivo,” Bio/Technology9:266-271; Co, M. S. et al. (1991) “Humanized Antibodies For AntiviralTherapy,” Proc. Natl. Acad. Sci. (U.S.A.) 88:2869-2873; Carter, P. etal. (1992) “Humanization Of An Anti-p185her2 Antibody For Human CancerTherapy,” Proc. Natl. Acad. Sci. (U.S.A.) 89:4285-4289; and Co, M. S. etal. (1992) “Chimeric And Humanized Antibodies With Specificity For TheCD33 Antigen,” J. Immunol. 148:1149-1154. In some embodiments, humanizedantibodies preserve all CDR sequences (for example, a humanized mouseantibody which contains all six CDRs from the mouse antibodies). Inother embodiments, humanized antibodies have one or more CDRs (one, two,three, four, five, six) which are altered with respect to the originalantibody, which are also termed one or more CDRs “derived from” one ormore CDRs from the original antibody. The ability to humanize an antigenis well known (see, e.g., U.S. Pat. Nos. 5,225,539; 5,530,101;5,585,089; 5,859,205; 6,407,213; 6,881,557).

The term “antibody “XX” is intended to denote an antibody orepitope-binding fragment thereof (for example antibody “C10-2”),comprising or consisting of the Light Chain, the Light Chain Variabledomain, or the Light Chain Variable domain CDR1-3, as defined by itsrespective SEQ ID NO, and the Heavy Chain, Heavy Chain Variable Domain,or Heavy Chain Variable Domain CDR1-3 as defined by its respective SEQID NO. In certain embodiments the antibody or epitope-binding fragmentthereof are defined by their entire Heavy Chain Variable Domaincomprising as defined by their SEQ ID NO and their Light Chain VariableDomain as defined by their SEQ ID NO.

Unless otherwise specified herein, numbering of amino acid residues inthe Fc region or constant domain of an antibody is according to the EUnumbering system, also called the EU index, as described in Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991.

As used herein, an antibody or an epitope-binding fragment thereof issaid to “specifically” bind a region of another molecule (i.e., anepitope) if it reacts or associates more frequently, more rapidly, withgreater duration and/or with greater affinity or avidity with thatepitope relative to alternative epitopes. It is also understood byreading this definition that, for example, an antibody orepitope-binding fragment thereof that specifically binds to a firsttarget may or may not specifically or preferentially bind to a secondtarget. As used herein, the term “binding” in the context of the bindingof an antibody to a predetermined antigen typically refers to bindingwith an affinity corresponding to a KD of about 10⁻⁷ M or less, such asabout 10⁻⁸ M or less, such as about 10⁻⁹ M or less when determined byfor instance surface plasmon resonance (SPR) technology in a BIAcore®3000 instrument using the antigen as the ligand and the antibody as theanalyte, and binds to the predetermined antigen with an affinitycorresponding to a KD that is at least ten-fold lower, such as at least100 fold lower, for instance at least 1,000 fold lower, such as at least10,000 fold lower, for instance at least 100,000 fold lower than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen. The amountwith which the affinity is lower is dependent on the KD of the antibody,so that when the KD of the antibody is very low (that is, the antibodyis highly specific), then the amount with which the affinity for theantigen is lower than the affinity for a non-specific antigen may be atleast 10,000 fold.

The term “kd” (sec−1 or 1/s), as used herein, refers to the dissociationrate constant of a particular antibody-antigen interaction. Said valueis also referred to as the koff value.

The term “ka” (M−1×sec−1 or 1/Msec), as used herein, refers to theassociation rate constant of a particular antibody-antigen interaction.

The term “KD” (M), as used herein, refers to the dissociationequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing the kd by the ka.

The term “KA” (M−1 or 1/M), as used herein, refers to the associationequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing the ka by the kd.

In one embodiment, the invention relates to an anti-tau antibody, orepitope-binding fragment thereof, which exhibits one or more of thefollowing properties:

-   -   (i) a substantial inability to bind to non-phosphorylated tau;    -   (ii) a substantial inability to bind to tau that is        phosphorylated at S404 and not phosphorylated at S396;    -   (iii) the ability to bind to tau phosphorylated at S396;    -   (iv) the ability to bind to tau phosphorylated at both S396 and        at S404;    -   (v) the ability to selectively discriminate between        phosphorylated tau residues S396 and S404 such that it is        substantially unable to bind the phosphorylated 404 residue        (pS404);    -   (vi) the ability to bind hyper-phosphorylated tau from human        Alzheimer's disease brains;    -   (vii) the ability to discriminate between pathological and        non-pathological human tau protein; and/or    -   (viii) the capability, when used as described herein with        immune-depleted rTg4510 extracts from transgenic mice, to        specifically reduce the hyperphosphorylated tau 64 kDa and 70        kDa bands by at least 90%, while not reducing the 55 kDa tau        band by more than 10%; or the capability, when used as described        herein with extracts from human AD post-mortem brains to        specifically reduce the S396 phosphorylated hyperphosphorylated        tau bands by at least 90%, while not reducing the        non-hyperphosphorylated tau bands by more than 10%.

A further embodiment of the invention relates to an antibody generatedby a method for generating high specificity, high affinity antibodiesthat are immunospecific for pathogenic hyperphosphorylated taucomprising residue a phosphorylated S396, wherein said method comprisesthe steps of:

-   -   (A) injecting an immunogen into a mammal, said immunogen        comprising the bi-phosphorylated peptide comprising 18-40, such        as at 18-30, such as 20-30 amino consecutive acid residues        comprising TDHGAEIVYK^((p))SPVVSGDT^((p))SPRHL (SEQ ID NO:37)        covering residues 386-410 of 2N4R tau, to thereby immunize said        mammal;    -   (B) repeating said immunization of said mammal two or more        times;    -   (C) screening a serum sample from said repeatedly immunized        mammal for the presence of high specificity, high affinity        antibodies capable of binding pathogenic hyperphosphorylated tau        comprising residue a phosphorylated S396, but substantially less        capable of binding non-pathogenic tau; and    -   (D) recovering said high specificity, high affinity antibodies.

As used herein, a “substantial inability” to bind a tau molecule denotesmore than a 20% difference, more than a 40% difference, more than a 60%difference, more than an 80% difference, more than a 100% difference,more than a 150% difference, more than a 2-fold difference, more than a4-fold difference, more than a 5-fold difference, or more than a 10-folddifference in functionality, relative to the detectable binding mediatedby a reference antibody.

The term “selective” and “immunoselective” when referring to the bindingcapabilities of an anti-tau antibody with respect to two epitopes isintended to denote that the observed binding under saturating conditionsexhibits at least an 80% difference, at least a 95% difference, and mostpreferably a 100% difference (i.e., no detectable binding to oneepitope). The term “selective” and “immunoselective” when referring to atau antibody is further intended to mean the antibody bindshyper-phosphorylated tau from human Alzheimer's disease brains and isable to discriminate between pathological and non-pathological human tauprotein.

The terms TBS-extractable (S1), high salt/sarkosyl-extractable (S3), andsarkosyl-insoluble (P3) fractions are fractions as obtained by the Taubiochemical fractionation described herein.

In some antibodies, only part of a CDR, namely the subset of CDRresidues required for binding, termed the SDRs, are needed to retainbinding in a humanized antibody. CDR residues not contacting therelevant epitope and not in the SDRs can be identified based on previousstudies (for example residues H60-H65 in CDR H2 are often not required),from regions of Kabat CDRs lying outside Chothia hypervariable loops(see, Kabat et al. (1992) SEQUENCES OF PROTEINS OF IMMUNOLOGICALINTEREST, National Institutes of Health Publication No. 91-3242;Chothia, C. et al. (1987) “Canonical Structures For The HypervariableRegions Of Immunoglobulins,” J. Mol. Biol. 196:901-917), by molecularmodeling and/or empirically, or as described in Gonzales, N. R. et al.(2004) “SDR Grafting Of A Murine Antibody Using Multiple Human GermlineTemplates To Minimize Its Immunogenicity,” Mol. Immunol. 41:863-872. Insuch humanized antibodies at positions in which one or more donor CDRresidues is absent or in which an entire donor CDR is omitted, the aminoacid occupying the position can be an amino acid occupying thecorresponding position (by Kabat numbering) in the acceptor antibodysequence. The number of such substitutions of acceptor for donor aminoacids in the CDRs to include reflects a balance of competingconsiderations. Such substitutions are potentially advantageous indecreasing the number of mouse amino acids in a humanized antibody andconsequently decreasing potential immunogenicity. However, substitutionscan also cause changes of affinity, and significant reductions inaffinity are preferably avoided. Positions for substitution within CDRsand amino acids to substitute can also be selected empirically.

The fact that a single amino acid alteration of a CDR residue can resultin loss of functional binding (Rudikoff, S. etc. (1982) “Single AminoAcid Substitution Altering Antigen-Binding Specificity,” Proc. Natl.Acad. Sci. (USA) 79(6):1979-1983) provides a means for systematicallyidentifying alternative functional CDR sequences. In one preferredmethod for obtaining such variant CDRs, a polynucleotide encoding theCDR is mutagenized (for example via random mutagenesis or by asite-directed method (e.g., polymerase chain-mediated amplification withprimers that encode the mutated locus)) to produce a CDR having asubstituted amino acid residue. By comparing the identity of therelevant residue in the original (functional) CDR sequence to theidentity of the substituted (non-functional) variant CDR sequence, theBLOSUM62.iij substitution score for that substitution can be identified.The BLOSUM system provides a matrix of amino acid substitutions createdby analyzing a database of sequences for trusted alignments (Eddy, S. R.(2004) “Where Did The BLOSUM62 Alignment Score Matrix Come From?,”Nature Biotech. 22(8):1035-1036; Henikoff, J. G. (1992) “Amino acidsubstitution matrices from protein blocks,” Proc. Natl. Acad. Sci. (USA)89:10915-10919; Karlin, S. et al. (1990) “Methods For Assessing TheStatistical Significance Of Molecular Sequence Features By Using GeneralScoring Schemes,” Proc. Natl. Acad. Sci. (USA) 87:2264-2268; Altschul,S. F. (1991) “Amino Acid Substitution Matrices From An InformationTheoretic Perspective,” J. Mol. Biol. 219, 555-565. Currently, the mostadvanced BLOSUM database is the BLOSUM62 database (BLOSUM62.iij). Table1 presents the BLOSUM62.iij substitution scores (the higher the scorethe more conservative the substitution and thus the more likely thesubstitution will not affect function). If an epitope-binding fragmentcomprising the resultant CDR fails to bind to tau, for example, then theBLOSUM62.iij substitution score is deemed to be insufficientlyconservative, and a new candidate substitution is selected and producedhaving a higher substitution score. Thus, for example, if the originalresidue was glutamate (E), and the non-functional substitute residue washistidine (H), then the BLOSUM62.iij substitution score will be 0, andmore conservative changes such as to aspartate, asparagine, glutamine,or lysine) are preferred.

TABLE 1 A R N D C Q E G H I L K M F P S T W Y V A +4 −1 −2 −2 0 −1 −1 0−2 −1 −1 −1 −1 −2 −1 +1 0 −3 −2 0 R −1 +5 0 −2 −3 +1 0 −2 0 −3 −2 +2 −1−3 −2 −1 −1 −3 −2 −3 N −2 0 +6 +1 −3 0 0 0 +1 −3 −3 0 −2 −3 −2 +1 0 −4−2 −3 D −2 −2 +1 +6 −3 0 +2 −1 −1 −3 −4 −1 −3 −3 −1 0 −1 −4 −3 −3 C 0 −3−3 −3 +9 −3 −4 −3 −3 −1 −1 −3 −1 −2 −3 −1 −1 −2 −2 −1 Q −1 +1 0 0 −3 +5+2 −2 0 −3 −2 +1 0 −3 −1 0 −1 −2 −1 −2 E −1 0 0 +2 −4 +2 +5 −2 0 −3 −3+1 −2 −3 −1 0 −1 −3 −2 −2 G 0 −2 0 −1 −3 −2 −2 +6 −2 −4 −4 −2 −3 −3 −2 0−2 −2 −3 −3 H −2 0 +1 −1 −3 0 0 −2 +8 −3 −3 −1 −2 −1 −2 −1 −2 −2 +2 −3 I−1 −3 −3 −3 −1 −3 −3 −4 −3 +4 +2 −3 +1 0 −3 −2 −1 −3 −1 +3 L −1 −2 −3 −4−1 −2 −3 −4 −3 +2 +4 −2 +2 0 −3 −2 −1 −2 −1 +1 K −1 +2 0 −1 −3 +1 +1 −2−1 −3 −2 +5 −1 −3 −1 0 −1 −3 −2 −2 M −1 −1 −2 −3 −1 0 −2 −3 −2 +1 +2 −1+5 0 −2 −1 −1 −1 −1 +1 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 +6 −4 −2 −2+1 +3 −1 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 +7 −1 −1 −4 −3 −2 S+1 −1 +1 0 −1 0 0 0 −1 −2 −2 0 −1 −2 −1 +4 +1 −3 −2 −2 T 0 −1 0 −1 −1 −1−1 −2 −2 −1 −1 −1 −1 −2 −1 +1 +5 −2 −2 0 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3−2 −3 −1 +1 −4 −3 −2 +11 +2 −3 Y −2 −2 −2 −3 −2 −1 −2 −3 +2 −1 −1 −2 −1+3 −3 −2 −2 +2 +7 −1 V 0 −3 −3 −3 −1 −2 −2 −3 −3 +3 +1 −2 +1 −1 −2 −2 0−3 −1 +4

The invention thus contemplates the use of random mutagenesis toidentify improved CDRs. In the context of the present invention,conservative substitutions may be defined by substitutions within theclasses of amino acids reflected n one or more of Tables 2, 3, or 4:

Amino Acid Residue Classes for Conservative Substitutions:

TABLE 2 Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg(R), and His (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn(N), and Gln (Q) Aliphatic Uncharged Residues Cly (G), Ala (A), Val (V),Leu (L), and Ile (I) Non-polar Uncharged Residues Cys (C), Met (M), andPro (P) Aromatic Residues Phe (F), Tyr (Y), and Trp (W)Alternative Conservative Amino Acid Residue Substitution Classes:

TABLE 3 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y WAlternative Physical and Functional Classifications of Amino AcidResidues:

TABLE 4 Alcohol Group-Containing Residues S and T Aliphatic Residues I,L, V and M Cycloalkenyl-Associated Residues F, H, W and Y HydrophobicResidues A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively ChargedResidues D and E Polar Residues C, D, E, H, K, N, Q, R, S and TPositively Charged Residues H, K and R Small Residues A, C, D, G, N, P,S, T and V Very Small Residues A, G and S Residues Involved In TurnFormation A, C, D, E, G, H, K, N, Q, R, S, P and T Flexible Residues Q,T, K, S, G, P, D, E and R

More conservative substitutions groupings include:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Additional groups of amino acids may also be formulated using theprinciples described in, e.g., Creighton (1984) Proteins: Structure andMolecular Properties (2d Ed. 1993), W. H. Freeman and Company.

Phage display technology can alternatively be used to increase (ordecrease) CDR affinity. This technology, referred to as affinitymaturation, employs mutagenesis or “CDR walking” and re-selection usesthe target antigen or an antigenic epitope-binding fragment thereof toidentify antibodies having CDRs that bind with higher (or lower)affinity to the antigen when compared with the initial or parentalantibody (See, e.g. Glaser et al. (1992) J. Immunology 149:3903).Mutagenizing entire codons rather than single nucleotides results in asemi-randomized repertoire of amino acid mutations. Libraries can beconstructed consisting of a pool of variant clones each of which differsby a single amino acid alteration in a single CDR and which containvariants representing each possible amino acid substitution for each CDRresidue. Mutants with increased (or decreased) binding affinity for theantigen can be screened by contacting the immobilized mutants withlabeled antigen. Any screening method known in the art can be used toidentify mutant antibodies with increased or decreased affinity to theantigen (e.g., ELISA) (See Wu et al. 1998, Proc. Nat. Acad. Sci.(U.S.A.) 95:6037; Yelton et al., 1995, J. Immunology 155:1994). CDRwalking which randomizes the Light Chain may be used possible (see,Schier et al., 1996, J. Mol. Bio. 263:551).

Methods for accomplishing such affinity maturation are described forexample in: Krause, J. C. et al. (2011) “An Insertion Mutation ThatDistorts Antibody Binding Site Architecture Enhances Function Of A HumanAntibody,” MBio. 2(1) pii: e00345-10. doi: 10.1128/mBio.00345-10; Kuan,C. T. et al. (2010) “Affinity-Matured Anti-Glycoprotein NMB RecombinantImmunotoxins Targeting Malignant Gliomas And Melanomas,” Int. J. Cancer10.1002/ijc.25645; Hackel, B. J. et al. (2010) “Stability And CDRComposition Biases Enrich Binder Functionality Landscapes,” J. Mol.Biol. 401(1):84-96; Montgomery, D. L. et al. (2009) “Affinity MaturationAnd Characterization Of A Human Monoclonal Antibody Against HIV-1 gp41,”MAbs 1(5):462-474; Gustchina, E. et al. (2009) “Affinity Maturation ByTargeted Diversification Of The CDR-H2 Loop Of A Monoclonal Fab DerivedFrom A Synthetic Naïve Human Antibody Library And Directed Against TheInternal Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fabs With ImprovedHIV-1 Neutralization Potency And Breadth,” Virology 393(1):112-119;Finlay, W. J. et al. (2009) “Affinity Maturation Of A Humanized RatAntibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A HighLevel Of Mutational Plasticity Both Inside And Outside TheComplementarity-Determining Regions,” J. Mol. Biol. 388(3):541-558;Bostrom, J. et al. (2009) “Improving Antibody Binding Affinity AndSpecificity For Therapeutic Development,” Methods Mol. Biol.525:353-376; Steidl, S. et al. (2008) “In Vitro Affinity Maturation OfHuman GM-CSF Antibodies By Targeted CDR-Diversification,” Mol. Immunol.46(1):135-144; and Barderas, R. et al. (2008) “Affinity Maturation OfAntibodies Assisted By In Silico Modeling,” Proc. Natl. Acad. Sci. (USA)105(26):9029-9034.

Thus, the sequence of CDR variants of encompassed antibodies or theirepitope-binding fragments may differ from the sequence of the CDR of theparent antibody, D1.2, C10-2, C5.2 or C8.3, through substitutions; forinstance substituted 4 amino acid residue, 3 amino acid residue, 2 aminoacid residue or 1 of the amino acid residues. According to an embodimentof the invention it is furthermore envisaged that the amino acids in theCDR regions may be substituted with conservative substitutions, asdefined in the 3 tables above. For example, the acidic residue Asp canbe substituted with Glu without substantially affecting the bindingcharacteristic of the antibody.

The term “normal tau” refers to normal brain tau containing 2-3 moles ofphosphate per mole of the protein.

The term “hyperphosphorylated tau” refers to a poly-phosphorylatedspecies of tau consistent with poly-anionic species induced mobilityshift in Western Blot or to a tau species which has more than five, sixor seven Serine, Threonine or Tyrosine sites phosphorylated.

The term “tau having residue 396 phosphorylated” relateshyperphosphorylated tau wherein residue 396 is phosphorylated andresidue 404 is or is not phosphorylated.

The term “transgenic non-human animal” refers to a non-human animalhaving a genome comprising one or more human heavy and/or light chaintransgenes or trans-chromosomes (either integrated or non-integratedinto the animal's natural genomic DNA) and which is capable ofexpressing fully humanized antibodies. For example, a transgenic mousecan have a humanized light chain transgene and either a humanized heavychain transgene or humanized heavy chain trans-chromosome, such that themouse produces humanized anti-tau antibody when immunized with tauantigen and/or cells expressing tau. The humanized heavy chain transgenemay be integrated into the chromosomal DNA of the mouse, as is the casefor transgenic mice, for instance HuMAb mice, such as HCo7 or HCol2mice, or the humanized heavy chain transgene may be maintainedextra-chromosomally, as is the case for trans-chromosomal KM mice asdescribed in WO02/43478. Such transgenic and trans-chromosomal mice(collectively referred to herein as “transgenic mice”) are capable ofproducing multiple isotypes of humanized monoclonal antibodies to agiven antigen (such as IgG, IgA, IgM, IgD and/or IgE) by undergoingV-D-J recombination and isotype switching.

Transgenic, nonhuman animal can also be used for production ofantibodies against a specific antigen by introducing genes encoding suchspecific antibody, for example by operatively linking the genes to agene which is expressed in the milk of the animal.

The term “treatment” or “treating” as used herein means ameliorating,slowing, attenuating, or reversing the progress or severity of a diseaseor disorder, or ameliorating, slowing, attenuating, or reversing one ormore symptoms or side effects of such disease or disorder. For purposesof this invention, “treatment” or “treating” further means an approachfor obtaining beneficial or desired clinical results, where “beneficialor desired clinical results” include, without limitation, alleviation ofa symptom, diminishment of the extent of a disorder or disease,stabilized (i.e., not worsening) disease or disorder state, delay orslowing of the progression a disease or disorder state, amelioration orpalliation of a disease or disorder state, and remission of a disease ordisorder, whether partial or total, detectable or undetectable.

An “effective amount,” when applied to an antibody or epitope-bindingfragment thereof of the invention, refers to an amount sufficient, atdosages and for periods of time necessary, to achieve an intendedbiological effect or a desired therapeutic result including, withoutlimitation, clinical results. The phrase “therapeutically effectiveamount,” when applied to an antibody or an epitope-binding fragmentthereof of the invention, is intended to denote an amount of theantibody, or epitope-binding fragment thereof, that is sufficient toameliorate, palliate, stabilize, reverse, slow, attenuate or delay theprogression of a disorder or disease state, or of a symptom of thedisorder or disease. In an embodiment, the method of the presentinvention provides for administration of the antibody, orepitope-binding fragment thereof, in combinations with other compounds.In such instances, the “effective amount” is the amount of thecombination sufficient to cause the intended biological effect.

A therapeutically effective amount of an anti-tau antibody orepitope-binding fragment thereof of the invention may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the anti-tau antibody, or epitope-bindingfragment thereof, to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antibody portion are outweighedby the therapeutically beneficial effects.

As indicated above, the present invention particularly relates tomonoclonal antibodies, or epitope-binding fragments thereof, and to acompletely new method for producing such molecules (and thus of suchepitope-binding fragments thereof). This method is outlined in FIG. 9.This ability of the new method to isolate monoclonal antibodies isexemplified herein by its use to isolate monoclonal antibodies that arecapable of specifically binding to the phosphorylated residue serine 396(^((p))S396) of human tau (SEQ ID NO:33). These antibodies are furthercharacterized by their ability to discriminate between phosphorylatedresidues serine 396 and serine 404 (pS404) such that they do not bind totau protein with phosphorylated serine 404 unless the tau is alsophosphorylated at residue 396.

The antibodies of the present invention, or epitope-binding fragmentthereof, have been generated and isolated by use of a novel a method(FIG. 9) which favors the selection of ^((p))S396 specific antibodies(FIG. 9). Furthermore, by applying this very strict antibody cloneselection procedure, antibodies have been obtained that are not onlyhighly specific towards S396, but also highly selective towards thephosphorylated ^((p))S396 epitope. These antibodies uniquely recognizetau from Alzheimer's disease brains. We also demonstrate that thescreening procedure outlined in FIG. 9 ensures the identification ofantibodies which possess a functional and therapeutic utility.

Antibodies were raised against the bi-phosphorylated peptide:TDHGAEIVYK^((p))SPVVSGDT^((p))SPRHL (SEQ ID NO:37) covering residues386-408 of 2N4R tau (Example 1). Mice were immunized with thephospho-peptide. Once sufficient antibody titres had been obtained, themice were sacrificed and hybridomas were generated (Example 2). Thehybridomas were screened using dot-blot (Example 3) and MSD ELISA withimmobilized human pathological and non-pathological tau (Example 4). Theability to discriminate between pathological and non-pathological humantau protein in dot-blot and Western blot was used for the selection ofhybridomas. Sixteen clones were selected, of which four hybridoma cloneswere recovered that produced antibodies which exhibited extraordinarycapabilities for binding to human pathological tau material.

Specific binding to pathological and non-pathological tau was alsodetermined by isolation of tau from diseased and non-diseased human ADbrains and immobilization of this material on MSD ELISA plates (Example4).

A further aspect of the invention relates to monoclonal antibody or anepitope-binding fragment thereof elicited against the bi-phosphorylatedpeptide comprising at least 18, such as at least 20 amino consecutiveacid residues within TDHGAEIVYK^((p))SPVVSGDT^((p))SPRHL (SEQ ID NO:37)covering residues 386-410 of 2N4R tau. In this aspect of the invention,the monoclonal antibody or an epitope-binding fragment thereof istypically elicited against the bi-phosphorylated peptide comprising18-40, such as at 18-30, such as 20-30 amino consecutive acid residuescomprising TDHGAEIVYK^((p))SPVVSGDT^((p))SPRHL (SEQ ID NO:37) coveringresidues 386-410 of 2N4R tau.

A further aspect of the invention is directed to the monoclonal antibodyor an epitope-binding fragment thereof of the invention, having aspecificity for phosphoTau (pTau) from AD-diseased patients overage-matched healthy controls, such that said monoclonal antibody or anepitope-binding fragment thereof has a specificity difference forphosphoTau (pTau) from AD-diseased patients over tau from age-healthymatched controls of more than 50-fold, such as more than 100-foldincrease in specificity for AD disease material compared to healthycontrol material in an ELISA based assay detect phosphoTau (pTau) inbrain homogenates from AD and from healthy control subjects, using aphospho- and multimer-specific Setup 1 ELISA as described herein.

A related aspect of the invention is directed to the monoclonal antibodyor an epitope-binding fragment thereof of the invention, having aspecificity for AD-diseased Tau such that said monoclonal antibody or anepitope-binding fragment thereof has a specificity difference for ADover age-healthy matched controls of more than 50-fold, such as morethan 100-fold increase in specificity for AD disease material comparedto healthy control material in an ELISA based assay detect phosphoTau(pTau) in brain homogenates from AD and from healthy control subjects,using a phospho- and multimer-specific Setup 1 ELISA.

Setup 1 ELISA method comprises the steps A) a capture of pathologicalhuman Tau antigens from AD brains using C10-2 coated plates; B)incubation of Tau antigens with increasing concentrations of pS396specific antibodies; and C) detection of the Tau antigen capture andantibody mediated inhibition using sulfo-tagged anti human (total) Tauantibodies from MSD.

The invention further relates to an antibody generated by a method forgenerating high specificity, high affinity antibodies that areimmunospecific for pathogenic hyperphosphorylated tau comprising residuea phosphorylated S396, wherein said method comprises the steps of:

(A) injecting an immunogen into a mammal, said immunogen comprising thebi-phosphorylated peptide comprising 18-40, such as at 18-30, such as20-30 amino consecutive acid residues comprisingTDHGAEIVYK^((p))SPVVSGDT^((p))SPRHL (SEQ ID NO:37) covering residues386-410 of 2N4R tau, to thereby immunize said mammal;(B) repeating said immunization of said mammal two or more times;(C) screening a serum sample from said repeatedly immunized mammal forthe presence of high specificity, high affinity antibodies capable ofbinding pathogenic hyperphosphorylated tau comprising residue aphosphorylated S396, but substantially less capable of bindingnon-pathogenic tau; and(D) recovering said high specificity, high affinity antibodies.

More specifically, step A comprises: coating of MSD plates (typicallyovernight at 4 C) with C10-2 antibody, typically 0.5 μg/ml (captureantibody) in coating buffer, blocking (typically 1 hour at roomtemperature) and washing, typically 3 times. Step B comprises: mixing ofsamples of P3 lysate (typically 1:1000=2-4 μg/ml total protein) and/orS1(p) (typically 1:300=20-40 ng/ml total protein) from AD (pooled from 3patients) with graded concentrations of pS396 peptide epitope specificantibody and incubating (typically 1 hour at room temperature). Thereactions are subsequently incubated for 2 hours on plates prepared instep A. Step C comprises detecting C10-2 captured Tau was usingsulfo-tagged human tau. Tau antibody (typically 1:50) from MSD followingmanufacturer instructions. Plates are analyzed on MSD SECTOR® S600. ADP3 and AD S1(p) are tested in a similar setup.

A further embodiment is directed to an antibody, or antigen-bindingfragment thereof, capable of immunospecifically binding to thephosphorylated residue 396 of human tau (SEQ ID NO:33), which has beenproduced or manufactured in a cell line such as a human cell line, amammal non-human cell line, an insect, yeast or bacterial cell line.

The antibody, or antigen binding fragment thereof, capable ofimmunospecifically binding to the phosphorylated residue 396 of humantau (SEQ ID NO:33), produced in a CHO cell line, HEK cell line, BHK-21cell line, murine cell line (such as a myeloma cell line), fibrosarcomacell line, PER.C6 cell line, HKB-11 cell line, CAP cell line and HuH-7human cell line.

Specific affinities and binding properties of D1.2 and C10-2 have beencharacterized using tau 386-410 (2R4N) peptides which are eitherphosphorylated or un-phosphorylated at position 396 or 404 (SEQ IDNOs:29-32). Using the specific immunization and screening protocol (FIG.9) outlined in this application will produce highly phosphor-serine-396(pS396) specific antibodies as demonstrated in FIG. 4.

In order to demonstrate that the antibodies are specific towardspathological tau, D1.2 and C10-2 antibodies have also been characterizedby immune-histochemical analysis (Example 6). The antibodies exhibithighly specific binding to neurofibrillary tangles in Alzheimer'sdisease brains and in sections from Tg4510 tau transgenic miceexpressing human (P301L) mutant tau (FIG. 5). No binding is observed totissue from human control brains and from non-transgenic mouse brains,demonstrating that the antibodies specifically bind human tau and inparticular tau associated with Alzheimer's pathology.

The unique capability of these antibodies to recognize tau associatedwith disease pathology is demonstrated here in Example 7. We compare thebinding of pathological vs. non-pathological tau in the assay describedin Example 3. The comparison is to five published tau antibodies:hACI-2B6, IPN002, HJ8.5, 2.10.3, and 4E4. FIG. 6 illustrates the bindingof each of the reference antibodies towards tau from healthy anddiseased brains, and binding to P301L human mutant tau isolated from 10month old Tg4510 tau transgenic mice. This demonstrates that theisolated antibodies exhibit an exceptionally high degree of specificityand selectivity towards human pathological tau. This selectivity issuperior to any of the comparator antibodies as shown in Table 5.

TABLE 5 mAb Tested AD/ctrl TG/wt hACl-2B6 3 1 IPN002 3 37 HJ8.5 3 51 4E4no binding 1 2.10.3 5 2 C5-2_C10-2 >100 118

At saturation binding antibodies D1.2 and C10-2 exhibit more than100-fold selectivity towards P3 tau isolated from human AD brains.

To demonstrate that the selected antibodies have functional andtherapeutic utility, antibodies were tested in in-vitro and in-celltau-aggregation assays (Example 8). These assays are functional assayswhich demonstrate that the antibodies are able to interfere with thepathological aggregation process of tau. HEK293 cells are transientlytransfected with human tau-P301L-FLAG (4R0N). Subsequently the cells areexposed to tau extracts from human AD brains or from transgenic Tg4510brains. This exposure to pathological tau promotes tau uptake into cellsand intracellular aggregation. Both immuno-depletion of tau-preparationsusing antibodies D1.2 and C10-2, and direct treatment of cells withthese antibodies is able to reduce the formation of tau aggregatesdramatically (FIG. 7).

Therapeutic utility of antibodies D1.2 and C10-2 has also been evaluatedin the human tau/PS1 mouse (Example 9). This mouse model is a more ADdisease relevant animal model which only generates AD pathology late inlife (12-18 month of age). However, the mice do exhibit tau hyperphosphorylation before the occurrence of solid tangle pathology. Micewere injected chronically for 13 weeks, twice weekly with 15 mg/kg dose.Antibody treated mice exhibit a dramatic reduction in phosphorylated tauas demonstrated in FIG. 9, indicating that chronic treatment withantibodies D1.2 and C10-2 will reduce tangle pathology and thussubsequent neurodegeneration in vivo.

The antibodies of the invention specifically remove hyperphosphorylatedTau from rTg4510 mouse brain extracts by immunodepletion methods.Moreover, the antibodies of the invention do not remove the normal Taufrom the homogenates, whereas the commercially available tau5 antibodydoes. In contrast to commercial antibodies which bind to tau proteinswherein phosphorylation at residue 404 or at both residues 404 and 396,the antibodies of the invention specifically remove thehyperphosphorylated tau by 95%, that is phosphorylated on serine 396.Experiments (Example 12) demonstrate that the antibody of the invention,despite only removing a very small fraction of the total tau in thebrain homogenate (8%), the antibodies do however specifically remove thehyperphosphorylated tau (by 90%). Accordingly, one aspect of theinvention is directed to a monoclonal antibody, or an epitope-bindingfragment thereof, capable of immunospecifically binding to thepathogenic hyperphosphorylated tau. Furthermore, in experiments whereinhyperphosphorylated Tau was removed using an antibody of the invention,the seeding activity is abolished. By removing hyperphosphorylated taufrom the homogenates, the homogenates no longer induce seeding of Taupathology. It has been proposed that reduction of seeding reduces thedevelopment of tangle formation and the progression of tauopathies,including Alzheimer's disease. Accordingly, a further aspect of theinvention is directed to an antibody of the invention for use in thereduction of the progression of AD or in the symptoms of AD.

More specifically, as detailed above, the invention relates to any oneof four monoclonal antibodies selected from the group comprising:

Antibody D1.2

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:1;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:2;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:3;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:4;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:5;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:6.

The antibody or epitope-binding fragment thereof may comprise or consistof the heavy chain variable domain of SEQ ID NO:8 and/or the light chainvariable domain of SEQ ID NO:7.

In a related embodiment, the antibody D1.2 or epitope-binding fragmentthereof may comprise or consist of the heavy chain of SEQ ID NO 8 and/orthe light chain of SEQ ID NO:34.

Antibody C10-2

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14.

The antibody or epitope-binding fragment thereof may comprise or consistof the heavy chain variable domain of SEQ ID NO:15 and/or the lightchain variable domain of SEQ ID NO:16.

The amino acid sequence of the heavy chain of humanized C10-2 antibodyis shown in SEQ ID NO:35. The amino acid sequence of the light chain ofhumanized C10-2 antibody is shown in SEQ ID NO:36.

Altogether, the Examples show that the antibodies of the invention,including C10-2, bind efficiently to AD-P3 antigens coated MSD plates.In comparison, commercial antibodies such as PHF-13, have low bindingactivity. Furthermore PHF-13 demonstrated substantial higher degree ofnon-specific binding in comparison to the antibodies of the invention(see Table 6 A-Table 6D). Table 6 shows that mC10-2 fluid phaseinhibition of Ptau antigen capture in C10-2 coated plate is effective(IC50=10-20 nM) whereas mD1.2 is ineffective (IC50=500-1000 nM). mC10-2fluid phase inhibition of p-tau antigen capture in mC10-2 coated plateis effective with an of IC50=10-20 nM) whereas PHF-13 is ineffective(IC50=500-1000 nM).

One aspect of the invention is directed to an antibody comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11.

A further aspect of the invention is directed to an antibody comprising

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14.

A further aspect of the invention is directed to an antibody comprising

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:12;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:13;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:14and one, two or three of

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:9;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:10;and

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:11.

Antibody C5.2

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:17;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:18;

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:19;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:20;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:21;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:22.

The antibody or epitope-binding fragment thereof may comprise or consistof the heavy chain variable domain of SEQ ID NO:23 and/or the lightchain variable domain of SEQ ID NO:24.

As can be seen from the crystal structure of FIG. 10, the epitope isbound across the heavy chain and light chains of C5.2. Accordingly, in arelated embodiment, the antibody of the invention or epitope-bindingfragment thereof comprises

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:17;or

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:18;or

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:19;and

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:20;or

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:21;or

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:22.

Antibody C8.3

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:25;

(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:26

(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:27;

(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:28;

(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:29;and

(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:30.

The antibody or epitope-binding fragment thereof may comprise or consistof the heavy chain variable domain of SEQ ID NO:31 and/or the lightchain variable domain of SEQ ID NO:32.

The antibody or epitope-binding fragment thereof is preferably a humanor humanized antibody.

The antibodies and epitope-binding fragment thereof mentioned above may,according to one embodiment, further comprise a variant of such lightand/or heavy chain CDR1, CDR2 or CDR3 (with no more than 4 amino aciddifferences, or no more than 3 amino acid differences, or no more than 2amino acid differences, or no more than 1 amino acid difference.

As can be seen from FIG. 11, HC CDR1, HC CDR2, HC CDR3 and LC CDR3 are,in at least one embodiment, important, for the binding to the 392-398region of Tau. In one embodiment of the invention, the antibody of theinvention, or epitope-binding fragment thereof comprises:

-   -   a) a Heavy Chain CDR1 comprising the amino acid sequence        selected from the group consisting of SEQ ID NO:4, SEQ ID NO:12,        SEQ ID NO:20, and SEQ ID NO:28;    -   b) a Heavy Chain CDR2 comprising the amino acid sequence        selected from the group consisting of SEQ ID NO:5, SEQ ID NO:13,        SEQ ID NO:21, and SEQ ID NO:29; and    -   c) a Heavy Chain CDR3 comprising the amino acid sequence        selected from the group consisting of SEQ ID NO:6, SEQ ID NO:14,        SEQ ID NO:22, and SEQ ID NO:30; and    -   d) a Light Chain CDR3 comprising the amino acid sequence        selected from the group consisting of SEQ ID NO:3, SEQ ID NO:11,        SEQ ID NO:19, and SEQ ID NO:27.

The antibody of the invention, or epitope-binding fragment thereof maycomprise

-   -   a) a Heavy Chain CDR1 comprising the amino acid sequence of SEQ        ID NO:20;    -   b) a Heavy Chain CDR2 comprising the amino acid sequence of SEQ        ID NO:21;    -   c) a Heavy Chain CDR3 comprising the amino acid sequence of SEQ        ID NO:22; and    -   d) a Light Chain CDR3 comprising the amino acid sequence of SEQ        ID NO:19.

In one aspect of the invention, the invention is directed to an antibodyor epitope-binding fragments thereof, that forms a hydrophobic pocketformed by L3:H3, L3:F8*, H1:H13, H2:Y1, H2:Y3 with Y394 of the taupeptide. In an embodiment, the invention is directed to an antibody thatcompetes with an antibody further described herein for forming ahydrogen bonding network between solvated ^((p))S396 and L3:T4, H1:R10,H1:T11, H3:R1, H3:T3; (*) L3:F8 is the C-terminal flanking frameworkresidue of CDR L3 (see FIG. 11).

As can be seen from the x-ray crystal structure, the antibody of theinvention binds with two levels of selectivity. The first level ofselectivity is selectivity for hyperphosphorylated pathological, tau andthe second level of selectivity is selectivity for a phosphorylatedserine residue wherein the phosphate of said phosphorylated serine ishydrogen bonded to the side chain of a tyrosine residue two residuesremoved from said phosphorylated serine. Accordingly, an interestingaspect of the invention is directed to an antibody or epitope-bindingfragment thereof selective for an amino acid motif ofhyperphosphorylated tau comprising of a phosphorylated serine tworesidues removed from a tyrosine residue. Typically, the amino acidmotif has the sequence:Y-X-S(phosphorylated)-P-wherein Y is tyrosine, X is a naturally occurring amino acid, P isproline and S(phosphorylated) is serine with a phosphorylated hydroxylside chain.

Similarly, an interesting aspect of the invention is directed to anantibody or epitope-binding fragment thereof which binds tophosphorylated tau, preferably hyperphosphorylated tau, wherein saidantibody or epitope-binding fragment thereof is selective for the aminoacid residue motif IA, wherein R is a side chain of a naturallyoccurring amino acid.

Without being bound to a particular theory, it is believed that theantibody of the invention is selective for amino acid motif IA when saidmotif is in a conformation adopted by pathological tau. Accordingly, theamino acid motif IA is typically the sequence selectively recognized bythe antibody of the invention. Accordingly, an interesting aspect of theinvention is directed to an antibody or epitope-binding fragment thereofwhich binds to phosphorylated tau, preferably hyperphosphorylated tau,wherein said antibody or epitope-binding fragment thereof is selectivefor the amino acid residue motif IB, wherein R is a side chain of anaturally occurring amino acid.

In a typical embodiment of this aspect of the invention, the inventionis directed to an antibody or epitope-binding fragment thereof whichbinds to phosphorylated tau, preferably hyperphosphorylated tau, whereinsaid antibody or epitope-binding fragment thereof is selective for theamino acid residue motif IB, wherein R is a side chain of a naturallyoccurring amino acid such as, but not limited to IC or ID.

The present invention also provides a method of reducing tau tangleformation in a patient, comprising administering to the patient in needof such treatment, a therapeutically effective amount of an antibody ofthe invention, or epitope-binding fragments thereof.

One aspect of the invention is directed to a method of treating ataupathy using an antibody of the invention, or epitope-bindingfragments thereof. Typically, the taupathy is selected from the groupconsisting of Alzheimer's disease, Argyrophilic Grain Disease (AGD),Psychosis, particularly Psychosis due to AD or Psychosis in patientswith AD, psychiatric symptoms of patients with Lewy body dementia,Progressive Supranuclear Palsy (PSP), Frontotemporal dementia (FTD orvariants thereof), TBI (traumatic brain injury, acute or chronic),Corticobasal Degeneration (CBD), Picks Disease, Primary age-relatedtauopathy (PART), Neurofibrillary tangle-predominant senile dementia,Dementia pugilistica, Chronic traumatic encephalopathy, stroke, strokerecovery, neurodegeneration in relation to Parkinson's disease,Parkinsonism linked to chromosome, Lytico-Bodig disease(Parkinson-dementia complex of Guam), Ganglioglioma and gangliocytoma,Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosingpanencephalitis, Huntington's disease, lead encephalopathy, tuberoussclerosis, Hallervorden-Spatz disease and lipofuscinosis. Moretypically, the taupathy is selected from the group consisting ofAlzheimer's disease, Argyrophilic Grain Disease (AGD), Psychosis,particularly Psychosis due to AD or Psychosis in patients with AD,psychiatric symptoms of patients with Lewy body dementia, ProgressiveSupranuclear Palsy (PSP), Frontotemporal dementia (FTD or variantsthereof), TBI (traumatic brain injury, acute or chronic), CorticobasalDegeneration (CBD), and Picks Disease. In particular, the tauopathiesmay be selected from Alzheimer's disease, Argyrophilic Grain Disease(AGD), Psychosis due to AD or Psychosis in patients with AD, andpsychiatric symptoms of patients with Lewy body dementia.

Accordingly, a further aspect of the invention is directed to anantibody of the invention or epitope-binding fragments thereof, for usein the treatment of a taupathy. Typically, the taupathy is selected fromthe group consisting of Alzheimer's disease, Argyrophilic Grain Disease(AGD), Psychosis, particularly Psychosis due to AD or Psychosis inpatients with AD, psychiatric symptoms of patients with Lewy bodydementia, Progressive Supranuclear Palsy (PSP), Frontotemporal dementia(FTD or variants thereof), TBI (traumatic brain injury, acute orchronic), Corticobasal Degeneration (CBD), Picks Disease, Primaryage-related tauopathy (PART), Neurofibrillary tangle-predominant seniledementia, Dementia pugilistica, Chronic traumatic encephalopathy,stroke, stroke recovery, neurodegeneration in relation to Parkinson'sdisease, Parkinsonism linked to chromosome, Lytico-Bodig disease(Parkinson-dementia complex of Guam), Ganglioglioma and gangliocytoma,Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosingpanencephalitis, Huntington's disease, lead encephalopathy, tuberoussclerosis, Hallervorden-Spatz disease and lipofuscinosis. Moretypically, the taupathy is selected from the group consisting ofAlzheimer's disease, Argyrophilic Grain Disease (AGD), Psychosis,particularly Psychosis due to AD or Psychosis in patients with AD,psychiatric symptoms of patients with Lewy body dementia, ProgressiveSupranuclear Palsy (PSP), Frontotemporal dementia (FTD or variantsthereof), TBI (traumatic brain injury, acute or chronic), CorticobasalDegeneration (CBD), and Picks Disease. In particular, the tauopathiesmay be selected from Alzheimer's disease, Argyrophilic Grain Disease(AGD), Psychosis due to AD or Psychosis in patients with AD, andpsychiatric symptoms of patients with Lewy body dementia.

A further aspect of the invention is directed to an antibody of theinvention or epitope-binding fragments thereof, in a compositiontogether with a pharmaceutically acceptable carrier, diluent, adjuvantand/or stabilizer. The antibodies of the invention, or epitope-bindingfragments thereof, may be used in therapy for the treatment of ataupathy. Typically, the taupathy is selected from the group consistingof Alzheimer's disease, Argyrophilic Grain Disease (AGD), Psychosis,particularly Psychosis due to AD or Psychosis in patients with AD,psychiatric symptoms of patients with Lewy body dementia, ProgressiveSupranuclear Palsy (PSP), Frontotemporal dementia (FTD or variantsthereof), TBI (traumatic brain injury, acute or chronic), CorticobasalDegeneration (CBD), Picks Disease, Primary age-related tauopathy (PART),Neurofibrillary tangle-predominant senile dementia, Dementiapugilistica, Chronic traumatic encephalopathy, stroke, stroke recovery,neurodegeneration in relation to Parkinson's disease, Parkinsonismlinked to chromosome, Lytico-Bodig disease (Parkinson-dementia complexof Guam), Ganglioglioma and gangliocytoma, Meningioangiomatosis,Postencephalitic parkinsonism, Subacute sclerosing panencephalitis,Huntington's disease, lead encephalopathy, tuberous sclerosis,Hallervorden-Spatz disease and lipofuscinosis. More typically, thetaupathy is selected from the group consisting of Alzheimer's disease,Argyrophilic Grain Disease (AGD), Psychosis, particularly Psychosis dueto AD or Psychosis in patients with AD, psychiatric symptoms of patientswith Lewy body dementia, Progressive Supranuclear Palsy (PSP),Frontotemporal dementia (FTD or variants thereof), TBI (traumatic braininjury, acute or chronic), Corticobasal Degeneration (CBD), and PicksDisease. In particular, the tauopathies may be selected from Alzheimer'sdisease, Argyrophilic Grain Disease (AGD), Psychosis due to AD orPsychosis in patients with AD, and psychiatric symptoms of patients withLewy body dementia.

The treatment envisioned by the present invention may be chronic and thepatient may be treated at least 2 weeks, such as at least for 1 month,6, months, 1 year or more.

The antibodies of the present invention may, for example, be monoclonalantibodies produced by the hybridoma method first described by Kohler etal., Nature 256, 495 (1975), or may be monoclonal antibodies produced byrecombinant DNA or other methods, or more preferably may be produced bythe novel method disclosed herein (FIG. 9). Monoclonal antibodies mayalso be isolated from phage antibody libraries using the techniquesdescribed in, for example, Clackson et al., Nature 352, 624-628 (1991)and Marks et al., J. Mol. Biol. 222, 581-597 (1991). Monoclonalantibodies may be obtained from any suitable source. Thus, for example,monoclonal antibodies may be obtained from hybridomas prepared frommurine splenic B lymphocyte cells obtained from mice immunized with anantigen of interest, for instance, in the form of cells expressing theantigen on the surface, or a nucleic acid encoding an antigen ofinterest. Monoclonal antibodies may also be obtained from hybridomasderived from antibody-expressing cells of immunized humans or fromnon-human mammals such as rats, rabbits, dogs, sheep, goats, primates,etc.

In one embodiment, the antibody of the invention is a humanizedantibody. Humanized monoclonal antibodies directed against tau may begenerated using transgenic or trans-chromosomal mice carrying parts ofthe human immune system rather than the mouse system. Such transgenicand transchromosomic mice include mice referred to herein as HuMAb(Humanized monoclonal antibody) mice and KM mice, respectively, and arecollectively referred to herein as “transgenic mice”.

The HuMAb mouse contains a human immunoglobulin gene mini-locus thatencodes un-rearranged human heavy variable and constant (p and Y) andlight variable and constant (K) chain immunoglobulin sequences, togetherwith targeted mutations that inactivate the endogenous μ and K chainloci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly, themice exhibit reduced expression of mouse IgM or K and in response toimmunization, the introduced human heavy and light chain transgenes,undergo class switching and somatic mutation to generate high affinityhuman IgG, κ monoclonal antibodies (Lonberg, N. et al. (1994), supra;reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113,49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 1365-93 (1995) and Harding, F. and Lonberg, N., Ann. N. Y. Acad. Sci 764536-546 (1995)). The preparation of HuMAb mice is described in detail inTaylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J.et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J.Immunol. 152, 2912-2920 (1994), Taylor, L. et al., InternationalImmunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology14, 845-851 (1996). See also U.S. Pat. Nos. 5,545,806, 5,569,825,5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318,5,874,299, 5,770,429, 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227,WO 92/22645, WO 92/03918 and WO 01/09187.

The HCo7, HCo12, HCo17 and HCo20 mice have a JKD disruption in theirendogenous light chain (kappa) genes (as described in Chen et al., EMBOJ. 12, 811-820 (1993)), a CMD disruption in their endogenous heavy chaingenes (as described in Example 1 of WO 01/14424), and a KCo5 human kappalight chain transgene (as described in Fishwild et al., NatureBiotechnology 14, 845-851 (1996)). Additionally, the HCo7 mice have aHCo7 human heavy chain transgene (as described in U.S. Pat. No.5,770,429), the HCo12 mice have a HCo12 human heavy chain transgene (asdescribed in Example 2 of WO 01/14424), the HCo17 mice have a HCo17human heavy chain transgene (as described in Example 2 of WO 01/09187)and the HCo20 mice have a HCo20 human heavy chain transgene. Theresulting mice express human immunoglobulin heavy and kappalight chaintransgenes in a background homozygous for disruption of the endogenousmouse heavy and kappa light chain loci.

In the KM mouse strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al., EMBO J. 12,811-820 (1993) and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187. Thismouse strain carries a human kappalight chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain transchromosomecomposed of chromosome 14 fragment hCF (SC20) as described in WO02/43478. HCo12-Balb/c, HCo17-Balb/c and HCo20-Balb/c mice can begenerated by crossing HCo12, HCo17 and HCo20 to KCo5[J/K](Balb) asdescribed in WO 09/097006.

The rTg4510 mouse is a known tauopathy model providing temporal andspatial control over mutant tau transgene expression. In the KM mousestrain, the endogenous mouse kappalight chain gene has been homozygouslydisrupted as described in Chen et al., EMBO J. 12, 811-820 (1993) andthe endogenous mouse heavy chain gene has been homozygously disrupted asdescribed in Example 1 of WO 01/09187. This mouse strain carries a humankappa light chain transgene, KCo5, as described in Fishwild et al.,Nature Biotechnology 14, 845-851 (1996). This mouse strain also carriesa human heavy chain trans-chromosome composed of chromosome 14epitope-binding fragment hCF (SC20) as described in WO 02/43478.

Splenocytes from these transgenic mice may be used to generatehybridomas that secrete humanized monoclonal antibodies according towell-known techniques. Humanized monoclonal or polyclonal antibodies ofthe present invention, or antibodies, of the present inventionoriginating from other species may also be generated transgenicallythrough the generation of another non-human mammal or plant that istransgenic for the immunoglobulin heavy and light chain sequences ofinterest and production of the antibody in a recoverable form therefrom.In connection with the transgenic production in mammals, antibodies maybe produced in, and recovered from, the milk of goats, cows, or othermammals. See for instance U.S. Pat. Nos. 5,827,690; 5,756,687; 5,750,172and 5,741,957.

The antibody of the invention may be of any isotype. The choice ofisotype typically will be guided by the desired effector functions, suchas ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4.Either of the human light chain constant domains, kappa or lambda, maybe used. If desired, the class of an anti-tau antibody of the presentinvention may be switched by known methods. For example, an antibody ofthe present invention that was originally IgM may be class switched toan IgG antibody of the present invention. Further, class switchingtechniques may be used to convert one IgG subclass to another, forinstance from IgGI to IgG2. Thus, the effector function of theantibodies of the present invention may be changed by isotype switchingto, e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody forvarious therapeutic uses. In one embodiment an antibody of the presentinvention is an IgG1 antibody, for instance an IgG1, K. An antibody issaid to be of a particular isotype if its amino acid sequence is mosthomologous to that isotype, relative to other isotypes.

In one embodiment, the antibody of the invention is a full-lengthantibody, preferably an IgG antibody, in particular an IgG1, K antibody.In another embodiment, the antibody of the invention is an antibodyepitope-binding fragment or a single-chain antibody.

Antibodies and epitope-binding fragments thereof may e.g. be obtained byepitope-binding fragmentation using conventional techniques, andepitope-binding fragments screened for utility in the same manner asdescribed herein for whole antibodies. For example, F(ab′)₂epitope-binding fragments may be generated by treating antibody withpepsin. The resulting F(ab′)₂ epitope-binding fragment may be treated toreduce disulfide bridges to produce Fab′ epitope-binding fragments. Fabepitope-binding fragments may be obtained by treating an IgG antibodywith papain; Fab′ epitope-binding fragments may be obtained with pepsindigestion of IgG antibody. An F(ab′) epitope-binding fragment may alsobe produced by binding Fab′-described below via a thioether bond or adisulfide bond. A Fab′ epitope-binding fragment is an antibodyepitope-binding fragment obtained by cutting a disulfide bond of thehinge domain of the F(ab′)₂. A Fab′-epitope-binding fragment may beobtained by treating an F(ab′)2 epitope-binding fragment with a reducingagent, such as dithiothreitol. Antibody epitope-binding fragment mayalso be generated by expression of nucleic acids encoding suchepitope-binding fragments in recombinant cells (see for instance Evanset al., J. Immunol. Meth. 184, 123-38 (1995)). For example, a chimericgene encoding a portion of an F(ab′)2 epitope-binding fragment couldinclude DNA sequences encoding the CH1 domain and hinge domain of the Hchain, followed by a translational stop codon to yield such a truncatedantibody epitope-binding fragment molecule.

In one embodiment, the anti-tau antibody is a monovalent antibody,preferably a monovalent antibody as described in WO2007059782 (which isincorporated herein by reference in its entirety) having a deletion ofthe hinge region. Accordingly, in one embodiment, the antibody is amonovalent antibody, wherein said anti-tau antibody is constructed by amethod comprising: i) providing a nucleic acid construct encoding thelight chain of said monovalent antibody, said construct comprising anucleotide sequence encoding the VL region of a selected antigenspecific anti-tau antibody and a nucleotide sequence encoding theconstant CL region of an Ig, wherein said nucleotide sequence encodingthe VL region of a selected antigen specific antibody and saidnucleotide sequence encoding the CL region of an Ig are operably linkedtogether, and wherein, in case of an IgG1 subtype, the nucleotidesequence encoding the CL region has been modified such that the CLregion does not contain any amino acids capable of forming disulfidebonds or covalent bonds with other peptides comprising an identicalamino acid sequence of the CL region in the presence of polyclonal humanIgG or when administered to an animal or human being; ii) providing anucleic acid construct encoding the heavy chain of said monovalentantibody, said construct comprising a nucleotide sequence encoding theVH region of a selected antigen specific antibody and a nucleotidesequence encoding a constant CH region of a human Ig, wherein thenucleotide sequence encoding the CH region has been modified such thatthe region corresponding to the hinge region and, as required by the Igsubtype, other regions of the CH region, such as the CH3 region, doesnot comprise any amino acid residues which participate in the formationof disulphide bonds or covalent or stable non-covalent inter-heavy chainbonds with other peptides comprising an identical amino acid sequence ofthe CH region of the human Ig in the presence of polyclonal human IgG orwhen administered to an animal human being, wherein said nucleotidesequence encoding the VH region of a selected antigen specific antibodyand said nucleotide sequence encoding the CH region of said Ig areoperably linked together; iii) providing a cell expression system forproducing said monovalent antibody; iv) producing said monovalentantibody by co-expressing the nucleic acid constructs of (i) and (ii) incells of the cell expression system of (iii).

Similarly, in one embodiment, the anti-tau antibody of the invention isa monovalent antibody, which comprises:

-   (i) a variable domain of an antibody of the invention as described    herein or an epitope-binding part of the said domain, and-   (ii) a CH domain of an immunoglobulin or a domain thereof comprising    the CH2 and CH3 domains, wherein the CH domain or domain thereof has    been modified such that the domain corresponding to the hinge domain    and, if the immunoglobulin is not an IgG4 subtype, other domains of    the CH domain, such as the CH3 domain, do not comprise any amino    acid residues, which are capable of forming disulfide bonds with an    identical CH domain or other covalent or stable non-covalent    inter-heavy chain bonds with an identical CH domain in the presence    of polyclonal human IgG.

In a further embodiment, the heavy chain of the monovalent antibody ofthe invention has been modified such that the entire hinge region hasbeen deleted.

In another further embodiment, the sequence of the monovalent antibodyhas been modified so that it does not comprise any acceptor sites forN-linked glycosylation.

The invention also includes “Bispecific Antibodies,” wherein an anti-taubinding region (e.g., a tau-binding region of an anti-tau monoclonalantibody) is part of a bivalent or polyvalent bispecific scaffold thattargets more than one epitope, (for example a second epitope couldcomprise an epitope of an active transport receptor, such that theBispecific Antibody would exhibit improved transcytosis across abiological barrier, such as the Blood Brain Barrier). Thus, in anotherfurther embodiment, the monovalent Fab of an anti-tau antibody may bejoined to an additional Fab or scfv that targets a different protein togenerate a bispecific antibody. A bispecific antibody can have a dualfunction, for example a therapeutic function imparted by an anti-taubinding domain and a transport function that can bind to a receptormolecule to enhance transfer cross a biological barrier, such as theblood brain barrier.

Antibodies and epitope-binding fragments thereof of the invention, alsoinclude single chain antibodies. Single chain antibodies are peptides inwhich the heavy and light chain Fv domains are connected. In oneembodiment, the present invention provides a single-chain Fv (scFv)wherein the heavy and light chains in the Fv of an anti-tau antibody ofthe present invention are joined with a flexible peptide linker(typically of about 10, 12, 15 or more amino acid residues) in a singlepeptide chain. Methods of producing such antibodies are described in forinstance U.S. Pat. No. 4,946,778, Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994), Bird et al., Science 242,423-426 (1988), Huston et al., PNAS USA 85, 5879-5883 (1988) andMcCafferty et al., Nature 348, 552-554 (1990). The single chain antibodymay be monovalent, if only a single VH and VL are used, bivalent, if twoVH and VL are used, or polyvalent, if more than two VH and VL are used.

The antibodies and epitope-binding fragments thereof described hereinmay be modified by inclusion may be modified by inclusion of anysuitable number of modified amino acids and/or associations with suchconjugated substituents. Suitability in this context is generallydetermined by the ability to at least substantially retain the tauselectivity and/or the tau specificity associated with thenon-derivatized parent anti-tau antibody. The inclusion of one or moremodified amino acids may be advantageous in, for example, increasingpolypeptide serum half-life, reducing polypeptide antigenicity, orincreasing polypeptide storage stability. Amino acid(s) are modified,for example, co-translationally or post-translationally duringrecombinant production (e.g., N-linked glycosylation at N-X-S/T motifsduring expression in mammalian cells) or modified by synthetic means.Non-limiting examples of a modified amino acid include a glycosylatedamino acid, a sulfated amino acid, a prenylated (e.g., farnesylated,geranyl-geranylated) amino acid, an acetylated amino acid, an acylatedamino acid, a PEGylated amino acid, a biotinylated amino acid, acarboxylated amino acid, a phosphorylated amino acid, and the like.References adequate to guide one of skill in the modification of aminoacids are replete throughout the literature. Example protocols are foundin Walker (1998) Protein Protocols On CD-Rom, Humana Press, Totowa, N.J.The modified amino acid may, for instance, be selected from aglycosylated amino acid, a PEGylated amino acid, a farnesylated aminoacid, an acetylated amino acid, a biotinylated amino acid, an amino acidconjugated to a lipid moiety, or an amino acid conjugated to an organicderivatizing agent.

The antibodies and epitope-binding fragments thereof of the invention,may also be chemically modified by covalent conjugation to a polymer tofor instance increase their circulating half-life. Exemplary polymers,and methods to attach them to peptides, are illustrated in for instanceU.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285 and 4,609,546. Additionalillustrative polymers include polyoxyethylated polyols and polyethyleneglycol (PEG) (e.g., a PEG with a molecular weight of between about 1,000and about 40,000, such as between about 2,000 and about 20,000, e.g.,about 3,000-12,000 g/mol).

The antibodies and epitope-binding fragments thereof of the presentinvention may further be used in a diagnostic method or as a diagnosticimaging ligand.

In one embodiment, antibodies and epitope-binding fragments thereof ofthe invention comprising one or more radiolabeled amino acids areprovided. A radiolabeled anti-tau antibody may be used for bothdiagnostic and therapeutic purposes (conjugation to radiolabeledmolecules is another possible feature). Non-limiting examples of suchlabels include, but are not limited to bismuth (²¹³Bi), carbon (¹¹C,¹³C, ¹⁴C), chromium (⁵¹Cr), cobalt (⁵⁷Co, ⁶⁰Co), copper (⁶⁴Cu),dysprosium (¹⁶⁵Dy), erbium (¹⁶⁹Er), fluorine (¹⁸F), gadolinium (¹⁵³Gd,¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga), germanium (⁶⁸Ge), gold (¹⁹⁸Au), holmium(¹⁶⁶Ho), hydrogen (³H), indium (¹¹¹In, ¹¹²n, ¹³¹In, ¹¹⁵In), iodine(¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), iridium (¹⁹²Ir), iron (⁵⁹Fe), krypton(^(81m)Kr), lanthanium (¹⁴⁰La), lutelium (¹⁷⁷Lu), manganese (⁵⁴Mn),molybdenum (⁹⁹Mo), nitrogen (¹³N, ¹⁵N), oxygen (¹⁵O), palladium (¹⁰³Pd),phosphorus (³²P), potassium (⁴²K), praseodymium (¹⁴²Pr), promethium(⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re), rhodium (¹⁰⁵Rh), rubidium (⁸¹Rb, ⁸²Rb),ruthenium (⁸²Ru, ⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc), selenium(⁷⁵Se), sodium (²⁴Na), strontium (⁸⁵Sr, ⁸⁹Sr, ⁹²Sr), sulfur (³⁵S),technetium (⁹⁹Tc), thallium (²⁰¹Tl), tin (¹³Sn, ¹⁷Sn), xenon (¹³³Xe),ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Yb), yttrium (⁹⁰Y), zinc (⁶⁵Zn) andzirconium (⁸⁹Zr). Zirconium (⁸⁹Zr) is particularly interesting. Methodsfor preparing radiolabeled amino acids and related peptide derivativesare known in the art (see for instance Junghans et al., in CancerChemotherapy and Biotherapy 655-686 (2nd edition, Chafner and Longo,eds., Lippincott Raven (1996)) and U.S. Pat. Nos. 4,681,581; 4,735,210;5,101,827; U.S. Pat. No. 5,102,990 (U.S. Pat. No. RE35,500), U.S. Pat.Nos. 5,648,471 and 5,697,902. For example, a radioisotope may beconjugated by a chloramine T method (Lindegren, S. et al. (1998)“Chloramine-T In High-Specific-Activity Radioiodination Of AntibodiesUsing N-Succinimidyl-3-(Trimethylstannyl)Benzoate As An Intermediate,”Nucl. Med. Biol. 25(7):659-665; Kurth, M. et al. (1993) “Site-SpecificConjugation Of A Radioiodinated Phenethylamine Derivative To AMonoclonal Antibody Results In Increased Radioactivity Localization InTumor,” J. Med. Chem. 36(9):1255-1261; Rea, D. W. et al. (1990)“Site-specifically radioiodinated antibody for targeting tumors,” CancerRes. 50(3 Suppl):857s-861s).

The invention also provides anti-tau antibodies and epitope-bindingfragments thereof that are detectably labeled using a fluorescent label(such as a rare earth chelate (e.g., a europium chelate)), afluorescein-type label (e.g., fluorescein, fluorescein isothiocyanate,5-carboxyfluorescein, 6-carboxy fluorescein, dichlorotriazinylaminefluorescein), a rhodamine-type label (e.g., ALEXA FLUOR® 568(Invitrogen), TAMRA® or dansyl chloride), VIVOTAG 680 XL FLUOROCHROME™(Perkin Elmer), phycoerythrin; umbelliferone, Lissamine; a cyanine; aphycoerythrin, Texas Red, BODIPY FL-SE® (Invitrogen) or an analoguethereof, all of which are suitable for optical detection.Chemiluminescent labels may be employed (e.g., luminol, luciferase,luciferin, and aequorin). Such diagnosis and detection can also beaccomplished by coupling the diagnostic molecule of the presentinvention to detectable substances including, but not limited to,various enzymes, enzymes including, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase, or to prosthetic group complexes such as, but notlimited to, streptavidin/biotin and avidin/biotin.

Chemiluminescent labels may be employed (e.g., luminol, luciferase,luciferin, and aequorin). Such diagnosis and detection can also beaccomplished by coupling the diagnostic molecule of the presentinvention to detectable substances including, but not limited to,various enzymes, enzymes including, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase, or to prosthetic group complexes such as, but notlimited to, streptavidin/biotin and avidin/biotin. Paramagnetic labelscan also be employed, and are preferably detected using PositronEmission Tomography (PET) or Single-Photon Emission Computed Tomography(SPECT). Such paramagnetic labels include, but are not limited tocompounds containing paramagnetic ions of Aluminum (Al), Barium (Ba),Calcium (Ca), Cerium (Ce), Dysprosium (Dy), Erbium (Er), Europium (Eu),Gandolinium (Gd), Holmium (Ho), Iridium (Ir), Lithium (Li), Magnesium(Mg), Manganese (Mn), Molybdenum (M), Neodymium (Nd), Osmium (Os),Oxygen (O), Palladium (Pd), Platinum (Pt), Rhodium (Rh), Ruthenium (Ru),Samarium (Sm), Sodium (Na), Strontium (Sr), Terbium (Tb), Thulium (Tm),Tin (Sn), Titanium (Ti), Tungsten (W), and Zirconium (Zi), andparticularly, Co⁺², CR⁺², Cr⁺³, Cu⁺², Fe⁺², Fe⁺³, Ga⁺³, Mn⁺³, Ni⁺²,Ti⁺³, V⁺³, and V⁺⁴, positron emitting metals using various positronemission tomographies, and non-radioactive paramagnetic metal ions.

Thus in one embodiment the anti-tau antibody or tau-binding fragmentthereof of the invention may be labelled with a fluorescent label, achemiluminescent label, a paramagnetic label, a radioisotopic label oran enzyme label. The labelled antibody of fragment may be used indetecting or measuring the presence or amount of said tau in the brainof a subject. This method may comprise the detection or measurement ofin vivo imaging of anti-tau antibody or tau-binding fragment bound tosaid tau and may comprises ex vivo imaging of said anti-tau antibody ortau-binding fragment bound to such tau.

In a further aspect, the invention relates to an expression vectorencoding one or more polypeptide chains of an antibody of the inventionor a tau-binding fragment thereof. Such expression vectors may be usedfor recombinant production of antibodies or epitope-binding fragmentsthereof of the invention.

An expression vector in the context of the present invention may be anysuitable DNA or RNA vector, including chromosomal, non-chromosomal, andsynthetic nucleic acid vectors (a nucleic acid sequence comprising asuitable set of expression control elements). Examples of such vectorsinclude derivatives of SV40, bacterial plasmids, phage DNA, baculovirus,yeast plasmids, vectors derived from combinations of plasmids and phageDNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, ananti-tau antibody-encoding nucleic acid is comprised in a naked DNA orRNA vector, including, for example, a linear expression element (asdescribed in, for instance, Sykes and Johnston, Nat Biotech 12, 355-59(1997)), a compacted nucleic acid vector (as described in for instanceU.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such aspBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleicacid vector (as described in, for instance, Schakowski et al., Mol Ther3, 793-800 (2001)), or as a precipitated nucleic acid vector construct,such as a CaPO₄-precipitated construct (as described in, for instance,WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigleret al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic CellGenetics 2, 603 (1981)). Such nucleic acid vectors and the usage thereofare well known in the art (see for instance U.S. Pat. Nos. 5,589,466 and5,973,972).

In one embodiment, the vector is suitable for expression of anti-tauantibodies or epitope-binding fragments thereof of the invention in abacterial cell. Examples of such vectors include expression vectors suchas BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J. Biol.Chem. 264, 5503-5509 (1989), pET vectors (Novagen, Madison, Wis.), andthe like.

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed.Current Protocols in Molecular Biology, Greene Publishing and WileyInterScience New York (1987), Grant et al., Methods in Enzymol 153,516-544 (1987), Mattanovich, D. et al. Methods Mol. Biol. 824, 329-358(2012), Celik, E. et al. Biotechnol. Adv. 30(5), 1108-1118 (2012), Li,P. et al. Appl. Biochem. Biotechnol. 142(2), 105-124 (2007), Böer, E. etal. Appl. Microbiol. Biotechnol. 77(3), 513-523 (2007), van der Vaart,J. M. Methods Mol. Biol. 178, 359-366 (2002), and Holliger, P. MethodsMol. Biol. 178, 349-357 (2002)).

In an expression vector of the invention, anti-tau antibody-encodingnucleic acids may comprise or be associated with any suitable promoter,enhancer, and other expression-facilitating elements. Examples of suchelements include strong expression promoters (e.g., human CMV IEpromoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTRpromoters), effective poly (A) termination sequences, an origin ofreplication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acids may also comprise an inducible promoter asopposed to a constitutive promoter such as CMV IE (the skilled artisanwill recognize that such terms are actually descriptors of a degree ofgene expression under certain conditions).

In an even further aspect, the invention relates to a recombinanteukaryotic or prokaryotic host cell, such as a transfectoma, whichproduces an antibody or epitope-binding fragment thereof of theinvention as defined herein or a bispecific molecule of the invention asdefined herein. Examples of host cells include yeast, bacteria, andmammalian cells, such as CHO or HEK cells. For example, in oneembodiment, the present invention provides a cell comprising a nucleicacid stably integrated into the cellular genome that comprises asequence coding for expression of an anti-tau antibody of the presentinvention or an epitope-binding fragment thereof. In another embodiment,the present invention provides a cell comprising a non-integratednucleic acid, such as a plasmid, cosmid, phagemid, or linear expressionelement, which comprises a sequence coding for expression of an anti-tauantibody or epitope-binding fragment thereof of the invention.

In a further aspect, the invention relates to a method for producing ananti-tau antibody of the invention, said method comprising the steps ofa) culturing a hybridoma or a host cell of the invention as describedherein above, and b) purifying the antibody of the invention from theculture media.

In one embodiment, the invention relates to a preparation that, as suchterm is used herein, comprises an anti-tau antibody as defined herein,and that is substantially free of naturally-arising antibodies that areeither not capable of binding to tau or that do not materially alter theanti-tau functionality of the preparation. Thus, such a preparation doesnot encompass naturally-arising serum, or a purified derivative of suchserum, that comprises a mixture of an anti-tau antibody and anotherantibody that does not alter the functionality of the anti-tau antibodyof the preparation, wherein such functionality is:

-   -   (i) a substantial inability to bind to non-phosphorylated tau;    -   (ii) a substantial inability to bind to tau that is        phosphorylated at S404 and not phosphorylated at S396;    -   (iii) the ability to bind to tau phosphorylated at S396;    -   (iv) the ability to bind to tau phosphorylated at both S396 and        at S404;    -   (v) the ability to selectively discriminate between        phosphorylated tau residues S396 and S404 such that it is        substantially unable to bind the phosphorylated 404 residue;    -   (vi) the ability to bind hyper-phosphorylated tau from human        Alzheimer's disease brains;    -   (vii) the ability to discriminate between pathological and        non-pathological human tau protein; and/or    -   (viii) the capability, when used as described herein with        immune-depleted rTg4510 extracts from transgenic mice, to        specifically reduce the hyperphosphorylated tau 64 kDa and 70        kDa bands by at least 90%, while not reducing the 55 kDa tau        band by more than 10% or the capability, when used as described        herein with extracts from human AD post-mortem brains, to        specifically reduce the S396 phosphorylated hyperphosphorylated        tau bands by at least 90%, while not reducing the        non-hyperphosphorylated tau bands by more than 10%.

The invention particularly relates to preparations of such an anti-tauantibody having a structural change in its amino acid sequence (in anyof its CDRs, variable domains, framework residues and/or constantdomains) relative to the structure of a naturally-occurring anti-tauantibody, wherein said structural change causes the anti-tau antibody toexhibit a markedly altered functionality (i.e., more than a 20%difference, more than a 40% difference, more than a 60% difference, morethan an 80% difference, more than a 100% difference, more than a 150%difference, more than a 2-fold difference, more than a 4-folddifference, more than a 5-fold difference, or more than a 10-folddifference in functionality) relative to the functionality exhibited bysaid naturally-occurring anti-tau antibody; wherein such functionalityis:

-   -   (i) a substantial inability to bind to non-phosphorylated tau;    -   (ii) a substantial inability to bind to tau that is        phosphorylated at S404 and not phosphorylated at S396;    -   (iii) the ability to bind to tau phosphorylated at S396;    -   (iv) the ability to bind to tau phosphorylated at both S396 and        at S404;    -   (v) the ability to selectively discriminate between        phosphorylated tau residues S396 and S404 such that it is        substantially unable to bind the phosphorylated 404 residue;    -   (vi) the ability to bind hyper-phosphorylated tau from human        Alzheimer's disease brains;    -   (vii) the ability to discriminate between pathological and        non-pathological human tau protein; and/or    -   (viii) the capability, when used as described herein with        immune-depleted rTg4510 extracts from transgenic mice, to        specifically reduce the hyperphosphorylated tau 64 kDa and 70        kDa bands by at least 90%, while not reducing the 55 kDa tau        band by more than 10%; or the capability, when used as described        herein with extracts from human AD post-mortem brains to        specifically reduce the S396 phosphorylated hyperphosphorylated        tau bands by at least 90%, while not reducing the        non-hyperphosphorylated tau bands by more than 10%.

The term “substantially free” of naturally-arising antibodies refers tothe complete absence of such naturally-arising antibodies in suchpreparations, or of the inclusion of a concentration of suchnaturally-arising antibodies in such preparations that does notmaterially affect the tau-binding properties of the preparations. Anantibody is said to be “isolated” if it has no naturally-arisingcounterpart or has been separated or purified from components whichnaturally accompany it.

The term “naturally-arising antibodies,” as it relates to suchpreparations, refers to antibodies (including naturally-arisingautoantibodies) elicited within living humans or other animals, as anatural consequence to the functioning of their immune systems.

Thus, the preparations of the present invention do not exclude, andindeed explicitly encompass, such preparations that contain an anti-tauantibody and a deliberately added additional antibody capable of bindingto an epitope that is not possessed by tau. Such preparationsparticularly include embodiments thereof wherein the preparationexhibits enhanced efficacy in treating Alzheimer's disease (AD),Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP),and Corticobasal Degeneration (CBD). Furthermore, the present inventionis directed to preparations that contain an anti-tau antibodyantibodies, or epitope-binding fragments thereof, intended for use inthe treatment of Psychosis, particularly Psychosis due to AD orPsychosis in patients with AD, and psychiatric symptoms of patients withLewy body dementia. Furthermore, the preparations of the presentinvention contain an anti-tau antibody antibodies, or epitope-bindingfragments thereof, that may be used in the treatment of stroke, strokerecovery, neurodegeneration in relation to Parkinson's disease.

In an even further aspect, the invention relates to a pharmaceuticalcomposition comprising:

-   -   (i) a tau antibody, or epitope-binding fragment thereof, both as        defined herein, or a preparation, as such term is defined        herein, that comprises such an anti-tau antibody or        epitope-binding fragment thereof; and    -   (ii) a pharmaceutically-acceptable carrier.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 22ndEdition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 2013.

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for the chosencompound of the present invention and the chosen mode of administration.Suitability for carriers and other components of pharmaceuticalcompositions is determined based on the lack of significant negativeimpact on the desired biological properties of the chosen compound orpharmaceutical composition of the present invention (e.g., less than asubstantial impact (10% or less relative inhibition, 5% or less relativeinhibition, etc.)) on epitope binding.

A pharmaceutical composition of the present invention may also includediluents, fillers, salts, buffers, detergents (e.g., a nonionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition. The diluent is selected to not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, or non-toxic, nontherapeutic, non-immunogenic stabilizers andthe like. The compositions may also include large, slowly metabolizedmacromolecules, such as proteins, polysaccharides like chitosan,polylactic acids, polyglycolic acids and copolymers (e.g., latexfunctionalized sepharose, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (e.g., oildroplets or liposomes).

The actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration. The selected dosage level will depend upon avariety of pharmacokinetic factors including the activity of theparticular compositions of the present invention employed, or the amidethereof, the route of administration, the time of administration, therate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

The pharmaceutical composition may be administered by any suitable routeand mode, including: parenteral, topical, oral or intranasal means forprophylactic and/or therapeutic treatment. In one embodiment, apharmaceutical composition of the present invention is administeredparenterally. The phrases “parenteral administration” and “administeredparenterally” as used herein means modes of administration other thanenteral and topical administration, usually by injection, and includeepidermal, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.

Additional suitable routes of administering a compound of the presentinvention in vivo and in vitro are well known in the art and may beselected by those of ordinary skill in the art.

In one embodiment that pharmaceutical composition is administered byintravenous or subcutaneous injection or infusion.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible with a compound of thepresent invention.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude water, saline, phosphate buffered saline, ethanol, dextrose,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organicesters, such as ethyl oleate, and/or various buffers. Other carriers arewell known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe present invention is contemplated.

Proper fluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention may also compriseisotonicity agents, such as sugars, polyalcohols, such as mannitol,sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Thecompounds of the present invention may be prepared with carriers thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Such carriers may include gelatin,glyceryl monostearate, glyceryl distearate, biodegradable, biocompatiblepolymers such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, collagen, polyorthoesters, and polylactic acid alone or with awax, or other materials well known in the art. Methods for thepreparation of such formulations are generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In one embodiment, the compounds of the present invention may beformulated to ensure proper distribution in vivo. Pharmaceuticallyacceptable carriers for parenteral administration include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the active compound, use thereof in thepharmaceutical compositions of the present invention is contemplated.Supplementary active compounds may also be incorporated into thecompositions.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, micro-emulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe an aqueous or non-aqueous solvent or dispersion medium containing forinstance water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. The proper fluidity may be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions may be brought about by including in thecomposition an agent that delays antibody absorption, for example,monostearate salts and gelatin. Sterile injectable solutions may beprepared by incorporating the active compound in the required amount inan appropriate solvent with one or a combination of ingredients e.g. asenumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients e.g. from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens in the above methods of treatment and uses describedherein are adjusted to provide the optimum desired response (e.g., atherapeutic response). For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. Parenteral compositions may be formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe present invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

The effective dosages and the dosage regimens for the antibodies orepitope-binding fragments thereof of the invention, depend on thedisease or condition to be treated and may be determined by personsskilled in the art. On any given day that a dosage is given, the dosagemay range from about 0.0001 to about 100 mg/kg, and more usually fromabout 0.01 to about 5 mg/kg, of the host body weight. For example,dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within therange of 1-10 mg/kg body weight. Exemplary dosages thus include: fromabout 0.1 to about 10 mg/kg/body weight, from about 0.1 to about 5mg/kg/body weight, from about 0.1 to about 2 mg/kg/body weight, fromabout 0.1 to about 1 mg/kg/body weight, for instance about 0.15mg/kg/body weight, about 0.2 mg/kg/body weight, about 0.5 mg/kg/bodyweight, about 1 mg/kg/body weight, about 1.5 mg/kg/body weight, about 2mg/kg/body weight, about 5 mg/kg/body weight, or about 10 mg/kg/bodyweight.

A physician having ordinary skill in the art may readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician could start doses of an antibody orepitope-binding fragment thereof of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a composition of the present invention will be that amountof the compound which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above. Administration may e.g. be intravenous,intramuscular, intraperitoneal, or subcutaneous. If desired, theeffective daily dose of a pharmaceutical composition may be administeredas two, three, four, five, six or more sub-doses administered separatelyat appropriate intervals throughout the day, optionally, in unit dosageforms. While it is possible for a compound of the present invention tobe administered alone, it is preferable to administer the compound as apharmaceutical composition as described above.

The labeled antibodies or epitope-binding fragments thereof of theinvention can be used for diagnostic purposes to detect, diagnose, ormonitor diseases or disorders. The invention provides for the detectionor diagnosis of a neurodegenerative or cognitive disease or disorder,including but not limited to Alzheimer's disease, Argyrophilic GrainDisease (AGD), Progressive Supranuclear Palsy (PSP), and CorticobasalDegeneration (CBD), comprising: (a) assaying the existence ofpyroglutamated As fragments in cells or tissue samples of a subjectusing one or more antibodies that specifically bind to tau; and (b)comparing the level of the antigen with a control level, e.g. levels innormal tissue samples, whereby an increase in the assayed level ofantigen compared to the control level of antigen is indicative of thedisease or disorder, or indicative of the severity of the disease ordisorder.

The antibodies or epitope-binding fragments thereof of the invention canbe used to assay tau or fragments of tau in a biological sample usingimmunohistochemical methods well-known in the art. Other antibody-basedmethods useful for detecting protein include immunoassays such as theenzyme linked immunoassay (ELISA) and the radioimmunoassay assay (RIA)and mesoscale discovery platform based assays (MSD). Suitable antibodylabels may be used in such kits and methods, and labels known in the artinclude enzyme labels, such as alkaline phosphatase and glucose oxidase;radioisotope labels, such as iodine (¹²⁵I, ¹³¹I), carbon (¹⁴C), sulfur(³⁵S), tritium (³H), indium (¹²¹In), and technetium (^(91m)Tc); andluminescent labels, such as luminol and luciferase; and fluorescentlabels, such as fluorescein and rhodamine.

The presence of labeled anti-tau antibodies or their tau-bindingfragments may be detected in vivo for diagnostic purposes. In oneembodiment, diagnosis comprises: a) administering to a subject aneffective amount of such labeled molecule; b) waiting for a timeinterval following administration to allow the labeled molecule toconcentrate at sites (if any) of Aβ deposition and to allow for unboundlabeled molecule to be cleared to background level; c) determining abackground level; and d) detecting the labeled molecule in the subject,such that detection of labeled molecule above the background level isindicative that the subject has the disease or disorder, or isindicative of the severity of the disease or disorder. In accordancewith such embodiment, the molecule is labeled with an imaging moietysuitable for detection using a particular imaging system known to thoseskilled in the art. Background levels may be determined by variousmethods known in the art, including comparing the amount of labeledantibody detected to a standard value previously determined for aparticular imaging system. Methods and systems that may be used in thediagnostic methods of the invention include, but are not limited to,computed tomography (CT), whole body scan such as positron emissiontomography (PET), magnetic resonance imaging (MRI), and sonography.

In a further aspect, the invention provides a monoclonal antibody, or anepitope-binding fragment thereof, as defined herein for use in therapy.

In a further aspect, the invention provides a monoclonal antibody, or anepitope-binding fragment thereof, as defined herein for use in treating,diagnosing or imaging of tauopathies.

In a further aspect, the invention provides a monoclonal antibody, or anepitope-binding fragment thereof, as defined herein for use in treatingAlzheimer's disease, Argyrophilic Grain Disease (AGD), ProgressiveSupranuclear Palsy (PSP), and Corticobasal Degeneration (CBD).

In a further aspect, the invention provides a monoclonal antibody, or anepitope-binding fragment thereof, as defined herein for use in themanufacture of a medicament for treating, diagnosing or imagingtauopathies.

Preferably, the medicament is for treating Alzheimer's disease (AD),Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP),and Corticobasal Degeneration (CBD) most preferably Alzheimer's disease(AD). The medicament is also preferably for the treatment of Psychosis,particularly Psychosis due to AD or Psychosis in patients with AD, andpsychiatric symptoms of patients with Lewy body dementia.

In a further aspect, the invention provides a method of treating,diagnosing or imaging Alzheimer's disease or other tauopathies in asubject, said method comprising administering the medicament monoclonalantibody or epitope-binding fragment thereof as defined herein, to saidsubject in an effective amount.

In a preferred embodiment, the treatment is chronic, preferably for atleast 2 weeks, such as at least for 1 month, 6, months, 1 year or more.

In a further aspect, the invention provides a kit comprising theantibody, or fragment thereof, as defined herein for use in therapy.

EMBODIMENTS

-   1. A monoclonal antibody, or an epitope-binding fragment thereof,    capable of immunospecifically binding to the phosphorylated residue    396 of human tau, such as phosphorylated residue 396 of SEQ ID    NO:33.-   2. The antibody according to embodiment 1 consisting of an intact    antibody.-   3. The antibody or epitope-binding fragment thereof according to    embodiment 1 or 2 comprising or consisting of an epitope-binding    fragment selected from the group consisting of: an Fv fragment (e.g.    single chain Fv and disulphide-bonded Fv); a Fab-like fragment (e.g.    Fab fragment, Fab′ fragment and F(ab)2 fragment); a mini-body    (Fv)2-CH3 domain, and a domain antibody (e.g. a single VH variable    domain or VL variable domain).-   4. The antibody or epitope-binding fragment thereof according to any    preceding embodiment, wherein the antibody is selected from the    group consisting of antibodies of subtype IgG1, IgG2, IgG3, or IgG4.-   5. The monoclonal antibody or epitope-binding fragment thereof    according to any of the previous embodiments which is human or    humanized.-   6. The monoclonal antibody, or epitope-binding fragment thereof,    according to any one of the preceding embodiments wherein the    antibody or epitope-binding fragment exhibits one or more of the    following properties    -   (a) selectivity and specificity for human pathological tau;    -   (b) a binding affinity (KD) for p-Tau 386-408 (pS396/pS404) (SEQ        ID NO:33) between 0.5-10 nM, such as 1-5 nM or 1-2 nM-   7. The monoclonal antibody, or epitope-binding fragment thereof,    according to any one of the preceding embodiments, wherein said    antibody does not substantially bind the phosphorylated 404 residue    on tau (SEQ ID NO:33).-   8. A monoclonal antibody, or an epitope-binding fragment thereof    comprising:    -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID        NO:1 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ ID        NO:2 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID        NO:3 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID        NO:4 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID        NO:5 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference; and    -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID        NO:6 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference.-   9. The monoclonal antibody according to embodiment 8, comprising the    heavy chain variable domain of SEQ ID NO:8 or an amino acid sequence    having no more than 4 amino acid differences, or no more than 3    amino acid differences, or no more than 2 amino acid differences, or    no more than 1 amino acid difference and/or the light chain variable    domain of SEQ ID NO:7, having no more than 4 amino acid differences,    or no more than 3 amino acid differences, or no more than 2 amino    acid differences, or no more than 1 amino acid difference.-   10. A monoclonal antibody, or an epitope-binding fragment thereof,    comprising:    -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID        NO:9 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ ID        NO:10 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID        NO:11 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID        NO:12 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID        NO:13 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference; and    -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID        NO:14 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference.-   11. The monoclonal antibody according to embodiment 10, comprising    the heavy chain variable domain of SEQ ID NO:16 or an amino acid    sequence having no more than 4 amino acid differences, or no more    than 3 amino acid differences, or no more than 2 amino acid    differences, or no more than 1 amino acid difference and or the    light chain variable domain of SEQ ID NO:15 or an amino acid    sequence having no more than 4 amino acid differences, or no more    than 3 amino acid differences, or no more than 2 amino acid    differences, or no more than 1 amino acid difference.-   12. A monoclonal antibody, wherein the epitope-binding fragment    comprises:    -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID        NO:17 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ ID        NO:18 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID        NO:19 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID        NO:20 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID        NO:21 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference; and    -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID        NO:22 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference.-   13. The monoclonal antibody according to embodiment 12, comprising    the heavy chain variable domain of SEQ ID NO:24 or an amino acid    sequence having no more than 4 amino acid differences, or no more    than 3 amino acid differences, or no more than 2 amino acid    differences, or no more than 1 amino acid difference and or the    light chain variable domain of SEQ ID NO:23 or an amino acid    sequence having no more than 4 amino acid differences, or no more    than 3 amino acid differences, or no more than 2 amino acid    differences, or no more than 1 amino acid difference.-   14. A monoclonal antibody, or an epitope-binding fragment thereof    comprising:    -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ ID        NO:25 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ ID        NO:26 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ ID        NO:27 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID        NO:28 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference;    -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID        NO:29 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference; and    -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID        NO:30 or an amino acid sequence having no more than 4 amino acid        differences, or no more than 3 amino acid differences, or no        more than 2 amino acid differences, or no more than 1 amino acid        difference.-   15. The monoclonal antibody according to embodiment 14, comprising    the heavy chain variable domain of SEQ ID NO:32 or an amino acid    sequence having no more than 4 amino acid differences, or no more    than 3 amino acid differences, or no more than 2 amino acid    differences, or no more than 1 amino acid difference and or the    light chain variable domain of SEQ ID NO:31 or an amino acid    sequence having no more than 4 amino acid differences, or no more    than 3 amino acid differences, or no more than 2 amino acid    differences, or no more than 1 amino acid difference.-   16. The antibody or epitope-binding fragment thereof according to    one of embodiments 1 to 7, wherein said antibody or fragment thereof    competes with the antibody or epitope-binding fragment thereof    defined in Embodiment 8-15 for binding to human tau.-   17. The antibody or epitope-binding fragment thereof according to    any preceding embodiment comprising an Fc domain.-   18. The antibody or epitope-binding fragment thereof according to    any preceding embodiment further comprising a moiety for increasing    in vivo half-life.-   19. The antibody or epitope-binding fragment thereof according to    Embodiment 18, wherein the moiety for increasing the in vivo    half-life is selected from the group consisting of polyethylene    glycol (PEG), human serum albumin, glycosylation groups, fatty acids    and dextran.-   20. The antibody or epitope-binding fragment thereof according to    any preceding embodiment wherein the antibody further comprises a    detectable moiety.-   21. The antibody or epitope-binding fragment thereof according to    Embodiment 20 wherein the detectable moiety is selected from the    group consisting of: a fluorescent label; a chemiluminescent label;    a paramagnetic label; a radio-isotopic label; or an enzyme label.-   22. The antibody or epitope-binding fragment thereof according to    Embodiments 20 or 21 wherein the detectable moiety comprises or    consists of a radioisotope.-   23. The antibody or epitope-binding fragment thereof according to    Embodiment 22 wherein the radioisotope is selected from the group    consisting of 99mTc, 111In, 67Ga, 68Ga, 72As, 89Zr, 1231 and 201TI.-   24. The antibody or epitope-binding fragment thereof according to    Embodiment 21 wherein the detectable moiety comprises or consists of    a paramagnetic isotope.-   25. The antibody or epitope-binding fragment thereof according to    Embodiment 24 wherein the paramagnetic isotope is selected from the    group consisting of 157Gd, 55Mn, 162Dy, 52Cr and 56Fe.-   26. The antibody or epitope-binding fragment thereof according to    any of Embodiments 20 to 25 wherein the detectable moiety is    detectable by an imaging technique such as SPECT, PET, MRI, optical    or ultrasound imaging.-   27. The antibody or epitope-binding fragment thereof according to    any of Embodiments 20 to 26 wherein the detectable moiety is joined    to the antibody or epitope-binding fragment thereof indirectly, via    a linking moiety.-   28. The antibody or epitope-binding fragment thereof according to    Embodiment 27 wherein the linking moiety is selected from the group    consisting of derivatives of    1,4,7,10-tetraazacyclododecane-1,4,7,10, tetraacetic acid (DOTA),    deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic    avid (DTPA), derivatives of    S-2-(4-Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic    acid (NOTA) and derivatives of    1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA).-   29. The monoclonal antibody, or epitope-binding fragment thereof    wherein the Heavy Chain is selected from the group consisting of SEQ    ID NO:8, SEQ ID NO:16, SEQ ID NO:24, SEQ ID NO:32, and SEQ ID NO:35,    and the Light Chain is selected from the group consisting of SEQ ID    NO:7, SEQ ID NO:15, SEQ ID NO:23, and SEQ ID NO:36-   30. A monoclonal antibody, or epitope-binding fragment thereof    comprising    -   (a) a Heavy Chain CDR1 comprising the amino acid sequence        selected from the group consisting of SEQ ID NO:4, SEQ ID NO:12,        SEQ ID NO:20, and SEQ ID NO:28;    -   (b) a Heavy Chain CDR2 comprising the amino acid sequence        selected from the group consisting of SEQ ID NO:5, SEQ ID NO:13,        SEQ ID NO:21, and SEQ ID NO:29; and    -   (c) a Heavy Chain CDR3 comprising the amino acid sequence        selected from the group consisting of SEQ ID NO:6, SEQ ID NO:14,        SEQ ID NO:22, and SEQ ID NO:30; and    -   (d) a Light Chain CDR3 comprising the amino acid sequence        selected from the group consisting of SEQ ID NO:3, SEQ ID NO:11,        SEQ ID NO:19, and SEQ ID NO:27.-   31. The antibody of the invention, or epitope-binding fragment    thereof according to any one of Embodiments 1 to 7 comprising    -   a) a Heavy Chain CDR1 comprising the amino acid sequence of SEQ        ID NO:20;    -   (b) a Heavy Chain CDR2 comprising the amino acid sequence of SEQ        ID NO:21;    -   (c) a Heavy Chain CDR3 comprising the amino acid sequence of SEQ        ID NO:22; and    -   (d) a Light Chain CDR3 comprising the amino acid sequence of SEQ        ID NO:19.-   32. An isolated nucleic acid molecule encoding an antibody or    epitope-binding fragment thereof as defined in any of Embodiments 1    to 31.-   33. A nucleic acid molecule according to Embodiment 32 wherein the    molecule is a cDNA molecule.-   34. A vector comprising a nucleic acid molecule as defined in    Embodiment 32 or 33.-   35. A recombinant host cell comprising a nucleic acid molecule as    defined in any of Embodiments 32 to 34.-   36. A method for producing an antibody or epitope-binding fragment    as defined in any of Embodiments 1 to 31, the method comprising    culturing a host cell as defined in Embodiment 35 under conditions    which permit expression of the encoded antibody or epitope-binding    fragment thereof.-   37. A preparation comprising the antibody or epitope-binding    fragment thereof according to any one of the previous claims,    wherein said preparation is substantially free of naturally-arising    antibodies that are either not capable of binding to tau or that do    not materially alter an anti-tau functionality of the preparation,    wherein said functionality is selected from the group consisting of:    -   (i) a substantial inability to bind to non-phosphorylated tau;    -   (ii) a substantial inability to bind to tau that is        phosphorylated at S404 and not phosphorylated at S396;    -   (iii) the ability to bind to tau phosphorylated at S396;    -   (iv) the ability to bind to tau phosphorylated at both S396 and        at S404;    -   (v) the ability to selectively discriminate between        phosphorylated tau residues S396 and S404 such that it is        substantially unable to bind the phosphorylated 404 residue;    -   (vi) the ability to bind hyper-phosphorylated tau from human        Alzheimer's disease brains;    -   (vii) the ability to discriminate between pathological and        non-pathological human tau protein; and/or    -   (viii) the capability, when used as described herein with        immune-depleted rTg4510 extracts from transgenic mice, to        specifically reduce the hyperphosphorylated tau 64 kDa and 70        kDa bands by at least 90%, while not reducing the 55 kDa tau        band by more than 10% %; or the capability, when used as        described herein with extracts from human AD post-mortem brains,        to specifically reduce the S396 phosphorylated        hyperphosphorylated tau bands by at least 90%, while not        reducing the non-hyperphosphorylated tau bands by more than 10%.-   38. A preparation comprising the antibody or epitope-binding    fragment thereof according to any one of the previous claims,    wherein said antibody or said epitope-binding fragment thereof    possesses a structural change in its amino acid sequence, relative    to the structure of a naturally-occurring anti-tau antibody, wherein    said structural change causes said antibody or said fragment to    exhibit an altered functionality relative to the functionality    exhibited by said naturally-occurring anti-tau antibody, wherein    said functionality is selected from the group consisting of:    -   (i) a substantial inability to bind to non-phosphorylated tau;    -   (ii) a substantial inability to bind to tau that is        phosphorylated at S404 and not phosphorylated at S396;    -   (iii) the ability to bind to tau phosphorylated at S396;    -   (iv) the ability to bind to tau phosphorylated at both S396 and        at S404;    -   (v) the ability to selectively discriminate between        phosphorylated tau residues S396 and S404 such that it is        substantially unable to bind the phosphorylated 404 residue;    -   (vi) the ability to bind hyper-phosphorylated tau from human        Alzheimer's disease brains;    -   (vii) the ability to discriminate between pathological and        non-pathological human tau protein; and/or    -   (viii) the capability, when used as described herein with        immune-depleted rTg4510 extracts from transgenic mice, to        specifically reduce the hyperphosphorylated tau 64 kDa and 70        kDa bands by at least 90%, while not reducing the 55 kDa tau        band by more than 10%; or the capability, when used as described        herein with extracts from human AD post-mortem brains, to        specifically reduce the S396 phosphorylated hyperphosphorylated        tau bands by at least 90%, while not reducing the        non-hyperphosphorylated tau bands by more than 10%.-   39. A pharmaceutical composition comprising the monoclonal antibody    or epitope-binding fragment thereof as defined in any of embodiments    1 to 31, or the preparation as defined in any of embodiments 37-38;    and a pharmaceutical acceptable carrier.-   40. The monoclonal antibody, or fragment thereof, of any of    embodiments 1-31, the preparation of any of embodiments 37-38, or    the pharmaceutical composition of embodiment 39, for use in    medicine.-   41. The monoclonal antibody, or fragment thereof, of any of    embodiments 1-31, the preparation of any of embodiments 37-38, or    the pharmaceutical composition of embodiment 39, for use in treating    a tauopathy.-   42. The monoclonal antibody, or fragment thereof, the preparation,    or the pharmaceutical composition, according to embodiment 41    wherein the tauopathy is selected from the group consisting of    Alzheimer's disease, Argyrophilic Grain Disease (AGD), Progressive    Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD),    Psychosis, particularly Psychosis due to AD or Psychosis in patients    with AD, and psychiatric symptoms of patients with Lewy body    dementia.-   43. Use of the monoclonal antibody, or fragment thereof, of any of    embodiments 1-31, the preparation of any of embodiments 37-38, or    the pharmaceutical composition of embodiment 39 in the manufacturing    of a medicament for treating a tauopathy.-   44. The use of the monoclonal antibody, or fragment thereof, the    preparation, or the pharmaceutical composition according to    embodiment 43 wherein the tauopathy is selected from the group    consisting of Alzheimer's disease, Argyrophilic Grain Disease (AGD),    Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration    (CBD, Psychosis due to AD or Psychosis in patients with AD, and    psychiatric symptoms of patients with Lewy body dementia.-   45. A method of treating Alzheimer's disease or other tauopathies in    a subject, said method comprising administering the monoclonal    antibody, or fragment thereof, of any of embodiments 1-31, the    preparation of any of embodiments 37-38, or the pharmaceutical    composition of embodiment 39 to said subject in an effective amount.-   46. The method according to embodiment 45, wherein the treatment is    chronic.-   47. The method according to embodiment 46, wherein the chronic    treatment is for at least 2 weeks, such as at least for 1 month, 6,    months, 1 year or more.-   48. The method according to any one of embodiments 45 to 47, wherein    the subject is human.-   49. A kit comprising the monoclonal antibody, or fragment thereof,    of any of embodiments 1-31, the preparation of any of embodiments    37-38, or the pharmaceutical composition of embodiment 39 for use in    medicine.-   50. The monoclonal antibody, or fragment thereof, of any of    embodiments 1-31, the preparation of any of embodiments 37-38, or    the pharmaceutical composition of embodiment 39 for use in detecting    or measuring the presence or amount of said tau in the brain of a    subject.-   51. The monoclonal antibody, or fragment thereof, the preparation or    the pharmaceutical composition of embodiment 50, wherein said    detection or measurement comprises in vivo imaging of said anti-tau    antibody bound to said tau.-   52. The monoclonal antibody, or fragment thereof, the preparation or    the pharmaceutical composition of embodiment 50, wherein said    detection or measurement comprises ex vivo imaging of said anti-tau    antibody or said fragment thereof, bound to said tau.-   53. A monoclonal antibody, or an epitope-binding fragment thereof,    capable of immunospecifically binding to the phosphorylated residue    396 of human tau (SEQ ID NO:33) in the presence of human tau    phosphorylated at residue 404 but not phosphorylated at residue 396.-   54. A monoclonal antibody or an epitope-binding fragment thereof    that exhibits immunospecifically binding to human tau comprising a    phosphorylated residue 396 according to the test criteria: i) the    antibody does not substantially bind to non-phosphorylated tau; ii)    the antibody does not substantially bind to tau phosphorylated at    404 when 396 is not phosphorylated; iii) the antibody does bind to    tau phosphorylated at 396; and iv) the antibody does bind to tau    when both 396 and 404 are phosphorylated.-   55. A monoclonal antibody, raised against the bi-phosphorylated    peptide: TDHGAEIVYK^((p))SPVVSGDT^((p))SPRHL (SEQ ID NO:37) covering    residues 386-410 of 2N4R tau, or an epitope-binding fragment    thereof, capable of immunospecifically binding to the phosphorylated    residue 396 of human tau (SEQ ID NO:33).-   56. The monoclonal antibody according to embodiment 55, wherein    hybridomas are screened with human pathological and non-pathological    tau to isolate clones that both i) were immunospecific towards the    either of the phospho-epitopes S396 and ii) specifically recognize    hyper-phosphorylated tau from human Alzheimer's disease brains,    wherein said antibodies are able to discriminate between    pathological and non-pathological human tau protein.-   57. A method of removing hyperphosphorylated Tau from a tangle said    tangle comprising hyperphosphorylated Tau said method comprising    contacting hyperphosphorylated Tau with an antibody, said antibody    selective for Tau having residue 396 phosphorylated, so as to result    the tangle being depleted of 90% of hyperphosphorylatedtau.-   58. A method of delaying the progression of Alzheimer's Disease in a    patient said method comprising reducing or attenuating the    accumulation of pathological tau protein in said patient, said    method comprising administering an antibody which removes a tau    protein with a phosphorylated 396 residue.-   59. A method of delaying the progression of Alzheimer's Disease in a    patient said method comprising removing the tau proteins that seed    for pathological tau proteins, wherein tau proteins having residue    396 phosphorylated are removed.-   60. A method of treating a patient with Alzheimer's Disease    comprising removing hyperphosphorylated Tau from a tangle said    tangle comprising hyperphosphorylated Tau and normal Tau by    contacting hyperphosphorylated Tau with an antibody selective for    Tau having residue 396 phosphorylated.-   61. A method according to any of embodiments 57 to 59 comprising the    use of an antibody as defined in any one of embodiments 1 to 31,    40-42 or 50 to 56.-   62. An isolated monoclonal antibody, or an isolated epitope-binding    fragment thereof, capable of immunospecifically binding to the    phosphorylated residue 396 of human tau (SEQ ID NO:33).-   63. A recombinant human or recombinant humanized monoclonal    antibody, or an isolated epitope-binding fragment thereof, capable    of immunospecifically binding to the phosphorylated residue 396 of    human tau (SEQ ID NO:33).-   64. A recombinant monoclonal antibody, or an epitope-binding    fragment thereof, raised against the bi-phosphorylated peptide:    TDHGAEIVYK^((p))SPVVSGDT^((p))SPRHL (SEQ ID NO:37) covering residues    386-410 of 2N4R tau, wherein said recombinant monoclonal antibody,    or an epitope-binding fragment thereof, is capable of    immunospecifically binding to the phosphorylated residue 396 of    human tau (SEQ ID NO:33).-   65. A pharmaceutical composition comprising an isolated monoclonal    antibody, or an isolated epitope-binding fragment thereof, wherein    said isolated monoclonal antibody, or an isolated epitope-binding    fragment thereof is as defined in any one of the above embodiments.-   66. A chimeric monoclonal antibody or an isolated epitope-binding    fragment thereof, capable of immunospecifically binding to the    phosphorylated residue 396 of human tau (SEQ ID NO:33).-   67. An antibody, or antigen-binding fragment thereof, as defined in    any of embodiments 1-31 and 51-56 which has been produced or    manufactured in a cell line such as a human cell line, a mammal    non-human cell line, an insect, yeast or bacterial cell line.-   68. The antibody, or antigen binding fragment thereof, according to    embodiment 67 produced in a CHO cell line, HEK cell line, BHK-21    cell line, murine cell line (such as a myeloma cell line),    fibrosarcoma cell line, PER.C6 cell line, HKB-11 cell line, CAP cell    line and HuH-7 human cell line.

EXAMPLES Example 1: Immunisation of Mice with Phospho-Peptides 396/404

C56/BL6 and FVB mice were immunised with 10 μg P30 conjugatedphosphorylated tau 386-408 (pS396/pS404) (SEQ ID NO:37) formulated inTiterMax adjuvant.

Mice (C56/BL6 and FVB strains, female and male. 2- to 3-month-old micewere immunized with peptide epitope P30 conjugated phosphorylated tau386-408.

Immunogenic P30 conjugated phosphorylated tau 386-408 (pS396/pS404)peptide was formulated in TiterMax (400 μg/ml peptide mixed 1:1 vol:vol)following the TiterMax/vendor protocol and mice were injectedsubcutaneously with 20 μg peptide (100 μl) of antigen. Control mice wereinjected with adjuvant only. All peptide-immunised mice were boostedwith 0.5 μg peptide/Titermax (10 μg/ml peptide formulated as describedabove and injected) at monthly intervals. The mice were finally boostedwith P30 conjugated phosphorylated tau 386-408 (pS396/pS404) withoutTitermax 3 days prior to fusion of splenocytes with SP-2 cells.Hybridomas were selected for re-cloning cycles after exhibiting positivebinding to ELISA plates that had been coated with 1 μg/ml phosphorylatedtau 386-408 (pS396/pS404), and exhibiting preferential binding activityto S1 and P3 antigens from AD and TG4510 brain lysate (described belowin Example 3). Such binding was compared with the binding activity ofsuch antibodies to brain lysate from controls, using dot blots and brainlysate coated ELISA or MSD plates.

Example 2: Hybridoma Generation

The mice were boosted with P30 conjugated phosphorylated tau 386-408(pS396/pS404) without Titermax 3 days prior to fusion of splenocyteswith SP-2 cells. Hybridomas were selected for re-cloning cycles afterpositive binding in ELISA plates coated with 1 μg/ml phosphorylated tau386-408 (pS396/pS404), and preferential binding activity to S1 and P3antigens from AD and TG4510 brain lysate in comparison to brain lysatefrom controls using dot blots and brain lysate coated ELISA or MSDplates.

Example 3 Western Blot and Dot-Blot Analysis of Specific Antibodies

Tau Biochemical Fractionation

Brain tissues from humans or rTg4510 mice overexpressing the human taumutation P301L were homogenized in 10 volumes of Tris-buffered salinecontaining protease and phosphatase inhibitors as follows: 50 mMTris/HCl (pH 7.4); 274 mM NaCl; 5 mM KCl; 1% protease inhibitor mixture(Roche); 1% phosphatase inhibitor cocktail I & II (Sigma); and 1 mMphenylmethylsulfonyl fluoride (PMSF; Sigma). The homogenates werecentrifuged at 27,000×g for 20 min at 4° C. to obtain supernatant (S1)and pellet fractions. Pellets were re-homogenized in 5 volumes of highsalt/sucrose buffer (0.8 M NaCl, 10% sucrose, 10 mM Tris/HCl, [pH 7.4],1 mM EGTA, 1 mM PMSF) and centrifuged as above. The supernatants werecollected and incubated with sarkosyl (1% final concentration; Sigma)for one hour at 37° C., followed by centrifugation at 150,000×g for onehour at 4° C. to obtain sarkosyl-insoluble pellets, referred to as P3fraction. The P3 pellet was resuspended in TE buffer (10 mM Tris/HCl [pH8.0], 1 mM EDTA) to a volume equivalent to half of the original volumeused for the brain homogenates.

Western and Dot Blots

Fractionated tissue extracts S1 and P3 were dissolved in SDS-samplebuffer containing 0.1 M DTT. The heat-treated samples (95° C. for 10min) were separated by gel electrophoresis on 4-12% Bis-Tris SDS-PAGEgels (Invitrogen) and transferred onto PVDF membranes (BioRadLaboratories, Hercules, Calif.). Dot blot samples were spotted directlyonto nitrocellulose membranes (Amersham, Pittsburgh, Pa.) at knownconcentrations across samples. Both Western and dot blot membranes wereblocked in 5% non-fat dry milk in TBS-Tween (0.5%) pH 7.4, followed byincubation in 1 μg/ml D1.2 or C10-2 overnight at 4° C. Membranes werewashed and incubated with peroxidase-conjugated anti-mouse IgG (1:5000;Jackson ImmunoResearch, West Grove, Pa.). Bound antibodies were detectedusing an enhanced chemiluminescence system (ECL PLUS kit; PerkinElmer).Quantitation and visual analysis of Western and dot blotimmunoreactivity was performed with a computer-linked LAS-4000BioImaging Analyzer System (Fujifilm, Tokyo, Japan) and Multi Gauge v3.1software (Fujifilm). Protein loading was adjusted by the volume oforiginal fractions and can be converted to original tissue wet weight.Results are shown in FIG. 1 and FIG. 2.

Example 4 Screening and Selection of 396/404 Antibodies UsingImmobilized Human Pathological Material

Hybridoma supernatants were screened for antibody binding in nunc platescoated with 1 μg/ml peptide phosphorylated tau 386-408 (pS396/pS404)using 0.1 M carbonate buffer pH 9.

Positive supernatants were subsequently diluted 1:50-1:800 in PBS, 0.1%BSA and 0.1 NP40 for binding in ELISA or MSD plates coated with brain(P3 pellet, see Example 3) lysate antigens from AD and healthy controls(HC), respectively. Brain lysate antigens were diluted 1500 fold in 0.1M Carbonate buffer pH9 prior to incubation/coating of ELISA or MSDplates. Wells were subsequently blocked 2 hrs at room temperature (PBS,3 mg/ml BSA, 0.1% NP-40) and antibody binding activity detected with HRP(DAKO) and sulfotag (MSD, product #) conjugated anti-mouse IgG followingvendor protocol. Selections of antibodies (D1-2, C5-2, C8-3 and C10-2)diluted in PBS with 0.1% BSA were characterised by dose response andshowed sub-nanomolar-nanomolar binding activity to AD-P3 antigen coatedplates were furthermore characterised for binding-activity to selectionof specific and control peptides. Results are shown in FIG. 3.

Example 5: Peptide Specificity and Binding Affinity

Antibodies positive for binding to pathological tau were furthercharacterised for apparent affinity (IC50) and selectivity/specificityto a range of phospho-peptide (p) epitopes. MSD plates were coated with100 ng/m phosphorylated tau 386-408 (pS396/pS404) as described above.Antibodies against phosphorylated tau were analysed in dose responseassays to identify antibody concentrations providing appropriateanalytical signal level (typical 5,000-20,000 RU in MSD corresponding to0.5-2% of maximal instrumental signal or OD signals of 1.0-1.5 at 450 nmin ELISA. A selection of antibodies was incubated with gradedconcentrations (0-1000 nM) of phosphorylated tau 386-408 (pS396/404) for2 hrs/room temperature. The reactions were subsequently applied topeptide coated MSD plates coated with 100 ng/ml peptide phosphorylatedtau 386-408 (pS396/pS404) as described above and binding activitymeasured. IC50 values from the inhibition assays correspond to apparentaffinities (KD) between 10-100 nM.

Specificity and phospho-selectivity: An appropriate concentration ofmonoclonal antibody was incubated with 100 nM double phosphorylated(pS396/pS404) non-phosphorylated or monophosphorylated (pS396 or pS404)phosphorylated tau 386-408 and analysed for binding activity (inhibitionassays). Control phosphorylated tau peptides (phosphorylated tau 260-270(pS262) or phosphorylated tau 240-270 (pS262) and recombinantnon-phosphorylated tau protein was analysed for comparison. All AD-P3antigen positive antibodies showed strong preference for phosphorylatedpeptide tau 386-408 (pS396/pS404) and monophosphorylated peptidephosphorylated tau 386-408 (pS396) and no binding activity formono-phosphorylated peptide phosphorylated tau 386-408 (pS404) andnon-phosphorylated peptide tau 386-408. Control phosphor-peptides tau240-270 and phosphorylated tau. Results are shown in FIG. 4 and FIG. 32.

Example 6: Histological Characterization of Antibodies byImmunohistochemistry

Mouse brain tissues were collected from 8 months old rTg4510 mice(overexpressing human P301L-tau under the CamII promoter) andnon-transgenic littermate (non-Tg), fixed in 4% paraformaldehyde andembedded in paraffin. Paraffin-embedded human brain samples of frontalcortex were acquired from Tissue Solutions (Glasgow, UK). Tissue fromdonors with diagnosed end stage Alzheimer's disease was compared toage-matched non-demented control donors. Four um thick sections weredeparaffinized and subjected to antigen retrieval by microwaving thesections in 10 mM Citrate buffer, pH 6, for 10 minutes. Endogenousperoxidases were blocked with 1% hydrogen peroxidase followed by 5%normal swine serum in PBS/1% BSA/0.3% Triton X-100 (PBS-BT). Sectionswere incubated overnight at 4° C. with D1.2 and C10-2 antibodies dilutedin PBS-BT at a range of concentrations. The sections were washed in PBS,0.25% BSA, 0.1% Triton X-100, before being incubated with a biotinylatedsecondary swine anti-mouse antibody (E0464; DAKO, Glostrup, Denmark) at1:500 for 1 hour. Following additional washing, StreptAvidin-BiotinComplex kit (Vector Laboratories, Burlingame, Calif.) was applied andimmunoreactivity was visualized with diaminobenzidine. Sections werecounterstained with hematoxylin. Results are shown in FIG. 5.

Example 7: Selectivity of Antibodies Towards Pathological Tau

MSD plates were coated with solubilized P3 antigens from AD brain(diluted 1:1500) or TG4510 brain (diluted 1:3000). Results are shown inFIG. 6.

Detection is performed as described in Example 4 above.

Example 8: HEK Cell Seeding Assay

EK293 cells were transiently transfected with human tau-P301L-FLAG in6-well plates 24 h after plating, followed 24 h later by incubation withbrain homogenate for 24 h, followed by splitting and replating cells andharvesting after an additional 24 h. Cells were lysed and sonicated inPBS, supplemented with 1% triton X, Phos-stop and complete phosphataseand protease inhibitors (Roche) buffer and ultracentrifugated at100,000×g for 30 min. The pellet was resuspended in SDS, sonicated andultracentrifugated for 30 min at 100,000×g. Supernatants were analyzedby western blotting. Cells expressing human tau-P301L showed insoluble(SDS fraction, E1/FLAG detection), hyperphosphorylated (D1.2)pS396detection) tau upon seeding with total brain homogenates from rTg4510tau transgenic mice. Cells treated with control brain homogenate frommice showed an absence of aggregated hyperphosphorylated human tau.Additionally, total cell lysates of HEK293 cells were analyzed using thetau aggregation assay from Cisbio. This assay is based on time-resolvedfluorescence using the same antibody for both donor (Tb3+ conjugated)and acceptor (d2 conjugated) antibody in FRET. A 10 μl sample was mixedwith 10 μl antibody mix and incubated for 20 h. The plate was read onthe Pherastar plate reader to assess time-resolved fluorescence (FRETsignal measured/integrated after switching of the excitation light). Theassay measures aggregated tau both in human autopsy material, rTg4510mice and in seeded HEK cells with high specificity and sensitivity.Results are shown in FIG. 7 and show that the seeding effect was notaffected by treatment with HEL, but was partially reversed by treatmentwith tau antibodies (C10-2>D1.2>hACI36-2B6-Ab1).

Example 9: Reversal of Functional (Electrophysiology (Elphys)) Responsein-Vivo for D1.2 and C10-2

In vivo electrophysiological assessment of synaptic transmission andplasticity in the CA1 area of the hippocampus in 4.5 to 5.5 months oldrTg4510 and tTA control mice showed that i) basal synaptic transmissionis significantly impaired in rTg4510 compared to tTA mice, and ii)paired-pulse facilitation is significantly reduced rTg4510 compared totTA mice.

All experiments were carried out in accordance with the EuropeanCommunities Council Directive (86/609/EEC) for the care and use oflaboratory animals and the Danish legislation regulating animalexperiments.

rTg4510 and tTA male mice (Taconic Europe A/S) aged 5 to 5.5 months wereused in the present study at the time of the recordings. Mice weregrouped-housed in controlled temperature (22±1.5° C.) and humidityconditions (55-65%) and kept in a 12:12 hour light/dark cycle (lights onat 0S:00h). Food and water were available ad libitum.

Animals were anesthetized with an intraperitoneal (i.p.) injection ofurethane (1.2 g/kg). Mice were then mounted in a stereotaxic frame,their temperature adjusted to 37.5° C. via a heating pad, and the skullwas exposed. A platinum wire was placed in the frontal bone to act as areference, and an additional hole was drilled for insertion of therecording and stimulating electrodes in the hippocampus, at thefollowing coordinates according to the atlas of Paxinos and Franklin(Paxinos and Franklin, 2001): recording, 1.5-1.7 mm posterior to Bregma,1.0-1.2 mm lateral to the midline, 1.4-1.7 mm below the surface of thebrain; stimulation, 1.8-2.0 mm posterior to Bregma, 1.5-1.7 mm lateralto the midline, 1.3-1.7 mm below the surface of the brain. Animals wereleft in the stereotaxic frame throughout the whole duration of therecordings and their level of anesthesia was regularly checked.

Field potentials (fEPSP) were evoked in the CA1 by electricalstimulation of the Schaffer collateral every 30 s, and the depth of therecording electrode was adjusted until a negative fEPSP was recorded inresponse to a unipolar square pulse. The slope of the evoked fEPSP wastypically measured between 30 and 70% of the maximum amplitude of thefEPSP.

Once an optimal fEPSP was induced, basal synaptic transmission wasassessed by the relationship between stimulation intensity and slope ofthe evoked fEPSP (input-output relationship). The different intensitiesof stimulation were 0, 25, 50, 75, 100, 150, 200, 300, 400, and 500 μA,and were applied successively in increasing order, with 2 to 3 repeatsat each intensity. Basal synaptic transmission was found to besignificantly impaired in rTg4510 compared to tTA mice.

Paired-pulse facilitation, a short-term synaptic plasticity believed torely on presynaptic mechanisms, was further measured in rTg4510 and tTAmice. Briefly, a pair of stimuli with an inter-stimulus interval (ISI)varying from 25 to 1000 ms was applied to the Schaffer collateral, andthe slope of the second fEPSP was compared to the slope of the firstfEPSP. Facilitation was observed at all ISIs, with a maximumfacilitation at ISIs of 50 and 75 ms. Interestingly, a significantlylower PPF was observed in rTg4510 mice when compared tTA mice.

The identified impairments in basal synaptic transmission andpaired-pulse facilitation in rTg4510 mice were further used as readoutto test antibody efficacy.

Recordings were performed in all experiments 2 to 4 days followingadministration of 4 doses of antibody twice per week for 2 weeks, i.p.).Basal synaptic transmission and paired-pulse facilitation were recordedin both hippocampi in each animal when possible, and further used asindividual experiments. Results are shown in FIG. 8 and show antibodyreversal of paired pulse facilitation and basal synaptic transmissiondeficits in CA1 evoked field potentials.

Example 10: Immunodepletion of Tau from rTg4510 Brain Extracts

60 μg mouse and humanized C10-2 antibody was immobilized to 300 μl ofMagnetic dynabead suspension (Immunoprecipitation Kit Dynabeads ProteinG Novex, Cat no 10007D). After thorough washing the beads were mixedwith 60 μl rTg4510 brain extract and incubated at room temperature for10 minutes. The magnetic beads were separated from the extract and theextracts were analysed by western blot. Depletion with mC10-2 and hC10-2removed tau aggregates 99 and 99.5% respectively. Results are shown inFIG. 12.

Example 11: HEK Cell Seeding Assay Using Immunodepleted Extracts

HEK293 cells were transiently transfected with human tau-P301L-FLAG in6-well plates. 24 h later cells were incubated with brain homogenatethat had been immunodepleted using humanized or mouse C10-2. After 24 hcells were re-plated and harvested after an additional 24 h. Cells werelysed and sonicated in TBS, supplemented with 1% triton X, phosphataseand protease inhibitors (Roche) and ultracentrifugated at 100,000×g for30 min. The pellet was resuspended in 1% SDS, sonicated andultracentrifugated for 30 min at 100,000×g. Supernatants were analyzedby western blotting. Cells expressing human tau-P301L showed insoluble(SDS fraction, E1/FLAG detection), hyperphosphorylated tau (D1.2/pS396Tau, running at a higher molecular weight) upon seeding with total brainhomogenates from rTg4510 tau transgenic mice. Cells treated with controlbrain homogenate from tTA mice showed an absence of aggregatedhyperphosphorylated human tau. Additionally, total cell lysates ofHEK293 cells were analyzed using the tau aggregation assay from Cisbio.Depletion with HEL and hHEL antibodies did not affect seeding, whereasdepletion with mC10-2 and hC10-2 prevented tau aggregation 88 and 96%and insoluble tau 97 and 100% respectively. Results are shown in FIG.13.

Example 11: Immunodepletion of Tau from rTg4510 Brain Extracts

100 μg mouse C10-2, D1.2 and Tau5 (Invitrogen) antibody was immobilizedto 500 μl of Magnetic dynabead suspension (Immunoprecipitation KitDynabeads Protein G Novex, Cat no 10007D). After thorough washing thebeads were mixed with 100 μl rTg4510 brain extract and incubated at roomtemperature for 10 minutes. The magnetic beads were separated from theextract and the extracts were analysed by western blot. C10-2 and D1.2do not remove the normal Tau from the homogenates, as the commerciallyavailable Tau5 antibody does. In contrast, two antibodies of theinvention specifically remove the hyperphosphorylated tau (64 kDa) by95%, that is tau phosphorylated on serine 396. Results are shown in FIG.14.

Example 12: Immunodepletion of Tau from Alzheimer's Brain Extracts

100 μg mouse C10-2 and D1 antibody was immobilized to 500 μl of Magneticdynabead suspension (Immunoprecipitation Kit Dynabeads Protein G Novex,Cat no 10007D). After thorough washing the beads were mixed with 100 μlAlzheimer brain extract and incubated at room temperature for 10minutes. The magnetic beads were separated from the extract and theextracts were analysed by western blot. D1.2 and C10-2 does only removea very small fraction of the total tau in the brain homogenate (8%). Theantibodies do however specifically remove the hyperphosphorylated tau(90%), specific for AD patients. Results are shown in FIG. 15.

Example 13: Seeding in rTg4510 Mice Using Immunodepleted Extracts

Transgenic mice expressing human mutated Tau (P301L 0N4R) under atet-off responsive element in CamK2 positive neurons (rTg4510) was used.This model normally starts developing Tau pathology at 3 months of age,but by feeding the mothers with doxycycline during pregnancy and for thefirst 3 weeks of the pup's life, the pathology develops at a later stage(starting after 6 months of age). The doxycycline pre-treated mice usedin the studies were 2.5 months old at the time-point of injection. Micewere anesthetized by isoflouran inhalation fixed in a stereotacticframe. The scull was exposed and adjusted until bregma and lambda was inlevel. A hole was drilled in the scull 2 mm lateral (right) and 2.4 mmposterior of the bregma. A 10 μl syringe beveled tip (SGE) was used toinject the seeding material 1.4 mm ventral to the brain surface at theat the above mentioned co-ordinates. 2 μl of the immunodepletedextracts, described in Examples 11 and 12, was slowly infused at thesite (1 μl/minute) and the syringe was left for 5 minutes beforeremoving it. The wound was closed by stiches and mice were heated whilewaking up. The mice were housed for 3 months and then sacrificed andperfusion fixed with 4% PFA.

Immunohistochemistry: Fixed brains were cut into 35 μm coronal sectionsat NSA and every 6^(th) section was stained for Tau tangles (Gallyassilver stain) and for hyperphosphorylated Tau (AT8). Positively stainedneurons (soma) were counted in ipsi- and contralateral sides ofhippocampi of all brains. All sub-regions of hippocampus were included.Eight sections were counted per brain. Results reflect the sum ofpositive neurons from the 8 sections. The background signal wasdetermined in 2 non-injected mice. By removing hyperphosphorylated taufrom the homogenates, the homogenates do no longer induce seeding of Taupathology. Results are shown in FIG. 16. Quantification of Tau pathologyin rTg4510 brains seeded with rTg4510 (A) or AD (B) brain homogenates.Prior to seeding the hyperphosphorylated Tau, but not normal Tau, hadbeen reduced in the homogenates by 90-95% by using C10-2 or D1.2. Byremoving hyperphosphorylated tau from the homogenates, the homogenatesno longer induce seeding of Tau pathology.

Homogenates from rTg4510 or Alzheimer brains can seed Tau pathology inrTg4510 mice at a stage when endogenous Tau pathology has not developed.By removing the hyperphosphorylated Tau from the homogenates by usingD1.2 or C10-2, as described in Examples 11 and 12. the seeding activityis abolished.

Example 14: Antibody Treatment in Seeded rTg4510 Mice

Doxycyclin treated rTg4510 mice (as described in Example 13) werechronically treated with mouse D1.2 or control antibody, 15 mg/kg/weekstarting at 2 months of age. At 2.5 months rTg4510 brain extract wasinfused into the hippocampus (as described in Example 13). Mice weresacrificed 1, 2 and 3 months after the brain infusion andimmunohistochemistry and the following analysis was performed asdescribed in Example 13. D1.2 treatment significantly reduced Taupathology 2 and 3 months after seeding had been initiated. Results areshown in FIG. 17.

Quantification of tangle bearing neurons in hippocampus of seededrTg4510 mice. The pathology increase with time and by treating the micewith D1.2 the pathology is significantly lower 2 and 3 months afterseeding. Quantification of tangle bearing neurons in hippocampus ofseeded rTg4510 mice. The pathology increase with time and by treatingthe mice with D1.2 the pathology is significantly lower 2 and 3 monthsafter seeding.

Homogenates from rTg4510 or Alzheimer brains can seed Tau pathology inrTg4510 mice at a stage when endogenous Tau pathology has not developed.By systemically treating the mice with D1.2 the development of taupathology can be significantly reduced.

Example 15: Immunodepletion of Tau from Alzheimer's Brain Extracts UsingHumanized Tau Antibodies

100 μg mouse and humanized C10-2 as well as prior art antibodies 2.10.3and HJ8.5 antibody (source) was immobilized to 500 μl of Magneticdynabead suspension (Immunoprecipitation Kit Dynabeads Protein G Novex,Cat no 10007D). After thorough washing the beads were mixed with 100 μlAlzheimer brain extract and incubated at room temperature for 10minutes. The magnetic beads were separated from the extract and theextracts were analysed by western blot and the CisBio assay. The mouseand humanized C10-2 efficiently removed 93 and 97% of the pS396phosphorylated Tau, but only 22 and 17% of the total Tau compared to thehHeI control antibody. The 2.10.3 antibody removed 91% of Serine 396phosphorylated tau and 10% of total tau. It seems like the 2.10.3 isless efficient in removing one of the hyper phosphorylated bands incomparison to the C10-2 antibodies (the middle 64 kDa band). The HJ8.5antibody has a completely different profile then both the C10-2 and2.10.3 antibodies, by removing the majority of tau, 89% of total tau and88% of pS396 Tau. Results are shown in FIGS. 23-24.

Example 16: Immunodepletion of Aggregated Tau from Alzheimer's BrainExtracts Using Humanized Tau Antibodies

The immunodepleted AD extracts described in Example 13 was also analysedby using the Tau aggregation assay described in example 10. The C10-2and HJ8.5 antibodies are more efficiently removing aggregated Tau fromthe AD material then the 2.10.3 antibody. In order of efficiency: HJ8.5(99%), hC10-2 (98%), mC10-2 (96%) and 2.10.3 (90%) all in comparison tohHeI antibody. Results are shown in FIG. 25.

Example 17: Immunodepletion of Tau

25 μg antibody (humanized C10-2 or 2.10.3) was immobilized to 125 μl ofMagnetic dynabead suspension (Immunoprecipitation Kit Dynabeads ProteinG Novex, Cat no 10007D). After thorough washing the coated beads weremixed with variable amounts of non-coated, washed beads. Starting from100% Ab coated beads, corresponding to 5 μg antibody, down to 100%non-coated beads. The total amount of beads was the same in all samples.The beads were mixed with 20 μl AD extract and incubated at roomtemperature for 10 minutes. The magnetic beads were separated from theextract and the extracts were aliquoted, snap frozen and kept at −80 Cuntil use.

Analysis of Depletion Using Western Blot

Samples were boiled in 1×LDS loading buffer and 100 mM DTT. A volumecorresponding to 3 μl of extracts were loaded on a 4-12% Bis-Tris NuPAGEGel (LifeTech Novex). After electrophoresis, the proteins were blottedover to a Immobilon-FL PVDF membrane (0.45 μm, IPFL10100, Millipore).The membrane was blocked with SEA blocking buffer (Prod #37527, Thermo).Tau and P-tau levels were assessed in the samples using Tau5 (Abcamab80579, 1:2000) mouse C10-2 (1 μg/ml), P-S199/202 (Invitrogen 44768 G,1:1000), P-S422 (Abcam ab79415, 1:750), human IPN (1 μg/ml). Gapdh andactin were used as a loading controls (Abcam ab9484, 1:20000, SigmaA5441, 1:20000). Secondary fluorophore conjugated IgG antibodies wasused (IRDye 800CW Goat anti-Human, IRDye 800CW, Goat anti-rabbit, IRDye680 Goat anti-mouse, LI-COR biosciences) and the signal was quantifiedusing Odyssey CLx and Image studio software (LI-COR biosciences).

Quantification of individual bands as well as signal in whole lanes wasdone and from this sigmoidal dose-response curves were plotted and whenpossible max effect and EC50 values were estimated.

Results

Both antibodies remove a small fraction of tau from the Alzheimer brainpreparation. 2.10.3, designed to have specificity for P-S422 tau removesup to 24% of the total tau amount, while C10-2 removes up to 15% of thetotal tau (see FIG. 26).

2.10.3 and C10-2 both remove more than 90% of the tau phosphorylated atSerine 422 although the amount of antibody needed to remove 50% of theP-S422 tau differ, for 2.10.3, 0.42 μg antibody is needed and for C10-2,0.27 μg is needed for the same effect (see FIG. 27).

C10-2 efficiently remove Tau being phosphorylated at serine 396 (Maxeffect: 88% and half of the effect is reached by using 0.30 μgantibody). 2.10.3 removes a smaller fraction of tau being phosphorylatedat the serine 396 (Max effect: 60% and half of that effect is reachedwhen using 0.63 μg antibody)(see FIG. 28). This indicates that all Taubeing phosphorylated at serine 422, also is phosphorylated at serine396, but that there is a portion of hyperphosphorylated tau beingphosphorylated at serine 396 where the phosphorylated serine at position422 is not present.

A large portion of the tau, being removed by C10-2, is alsophosphorylated at Serine 199/202, since 69% of the tau having thatphosphporylation is affected by the immunodepletion (50% of the effectwhen using 0.34 μg antibody)(see FIG. 29). The 2.10.3 immunodepletiondoes not give a sigmoidal dose response on the P-S199/202 tau although adrop in signal is seen with increasing amount of antibody (max 52%reduction when using the max amount of antibody (5 μg) (see FIG. 29).

This data indicates that the C10-2 antibody targeting the phosphorylatedserine 396 binds a larger pool of the hyperphosporylated tau then the2.10.3 antibody targeting the phosphorylated serine at the 422 position.

Example 18: Antibody Mediated Inhibition of mC10-2 Specific Capture ofPathological Tau Antigens in AD Brain Lysates

Materials and Methods

Material

Coating buffer: Carbonate buffer pH 8.5, 150 mM NaCL. Blocking buffer:3% BSA (fraction V), 0.1% NP40 in PBS pH7.4. Washing buffer: 0.1% BSA(fraction V), 0.1% NP40 in PBS, pH 7.4. Sulfotag goat total humanizedTau antibody (MSD D221LA-1, 50 μg/ml)

Method aim to measure capture of pathological human Tau antigens from ADbrains using C10-2 coated plates (step A) after incubation of Tauantigens with increasing concentrations of pS396 specific antibodies(step B). The Tau antigen capture and antibody mediated inhibition wasdetected using sulfo-tagged anti human (total) Tau antibodies from MSD

A: MSD plates were coated (o/n at 4C) with 0.5 μg/ml mC10-2 (captureantibody) in coating buffer and subsequently blocked for 1 hour at roomtemperature) and washed 3 times. (FIG. 30)

B: Samples P3 lysate (1:1000=2-4 μg/ml total protein) and/or S1(p)(1:300=20-40 ng/ml total protein) from AD (pool from 3 patient) weremixed with graded concentrations of pS396 peptide epitope specificantibody and incubated for 1 hour at room temperature. The reactionswere subsequently incubated 2 hours on plates prepared in step A. (FIG.31)

C: C10-2 captured Tau was detected using sulfo-tagged human tau. Tauantibody (1:50) from MSD following manufacture instruction. Plates wereanalyzed on MSD SECTOR® S 600. AD P3 and AD S1(p) was tested in similarsetup. (FIG. 33/34)

TABLE 6A mouse C10-2, + tau peptide, 10 μM mean Signal signal signalPBS/0.1% BSA 388 403 373 C10-2 3 ng/ml 366 384 348 C10-2 10 ng/ml 383398 367 C10-2 30 ng/ml 345 384 306 C10-2 100 ng/ml 357 401 313 C10-2 300ng/ml 407 434 379 C10-2 1000 ng/ml 451 462 439 C10-2 10000 ng/ml 870 920820

TABLE 6B mouse C10-2, + PBS/0.1% BSA mean Signal signal signal PBS/0.1%BSA + PBS 303 293 312 C10-2 3 ng/ml + PBS 1881 1890 1871 C10-2 10ng/ml + PBS 5721 5863 5579 C10-2 30 ng/ml + PBS 11922 12044 11799 C10-2100 ng/ml + PBS 21833 21925 21741 C10-2 300 ng/ml + PBS 30410 3031130508 C10-2 1000 ng/ml + PBS 38524 38233 38814 C10-2 10000 ng/ml + PBS51171 51253 51089

TABLE 6C mouse clone PHF 13, + tau peptide, 10 μM mean Signal signalsignal PBS/0.1% BSA 287 286 287 PHF 13 1000000 280 284 276 PHF 13 300000299 305 292 PHF 13 100000 355 370 340 PHF 13 30000 481 472 490 PHF 1310000M 953 1019 886 PHF 13 3000 2182 2279 2084 PHF13 1000 6896 7542 6249

TABLE 6D mouse clone PHF 13, ± PBS/0.1% BSA mean Signal signal signalPBS/0.1% BSA + PBS 281 282 280 PHF 13 1000000 + PBS 335 358 312 PHF 13300000 + PBS 560 568 551 PHF 13 100000 + PBS 852 856 847 PHF 13 30000 +PBS 1579 1661 1496 PHF 13 10000 + PBS 2882 2899 2864 PHF 13 3000 + PBS5792 6126 5458 PHF 13 1000 + PBS 12639 13654 11624Table 6A-6D:Tau antigen capture inhibition

The invention claimed is:
 1. An immunoglobulin molecule comprising: (a)a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:25; (b) aLight Chain CDR2 having the amino acid sequence of SEQ ID NO:26; (c) aLight Chain CDR3 having the amino acid sequence of SEQ ID NO:27; (d) aHeavy Chain CDR1 having the amino acid sequence of SEQ ID NO:28; (e) aHeavy Chain CDR2 having the amino acid sequence of SEQ ID NO:29; and (f)a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:30. 2.The immunoglobulin molecule according to claim 1, wherein saidimmunoglobulin molecule is a monoclonal antibody or a bispecificantibody having an Fc domain.
 3. The immunoglobulin molecule accordingto claim 1, wherein said immunoglobulin molecule is an epitope-bindingfragment of a monoclonal antibody that is selected from the groupconsisting of: an Fv fragment, an Fv fragment fused to an Fc domain, aFab fragment, a Fab′ fragment, a F(ab)2 fragment, a single VH variabledomain and a single VL variable domain.
 4. The immunoglobulin moleculeaccording to claim 1, wherein said immunoglobulin molecule is ahumanized antibody.
 5. The immunoglobulin molecule according to claim 1,comprising the heavy chain of SEQ ID NO:32 and/or the light chain of SEQID NO:31.
 6. A pharmaceutical composition comprising immunoglobulinmolecule according to claim 1, and a pharmaceutical acceptable carrier.7. A nucleic acid encoding the immunoglobulin molecule according toclaim
 1. 8. A method of treating Alzheimer's disease or other tauopathyin a subject in need thereof, said method comprising administering tosaid subject an effective amount of the immunoglobulin molecule ofclaim
 1. 9. A method of diagnosing or imaging Alzheimer's disease orother tauopathy in a subject in need thereof, wherein said methodcomprises administering to said subject an amount of the immunoglobulinmolecule of claim 1 sufficient to detect or measure the presence oramount of said hyperphosphorylated variant of human tau in the brain ofa said subject.
 10. A method of treating Alzheimer's Disease in asubject in need thereof, wherein said method comprises administering tosaid subject an amount of the immunoglobulin molecule of claim 1sufficient to reduce or attenuate the accumulation of pathological tauprotein in said patient.
 11. The immunoglobulin molecule of claim 1,wherein the immunoglobulin molecule is capable of immunospecificallybinding to the phosphorylated residue 396 of human tau (SEQ ID NO:33).